Methods and compositions for nucleic acid amplification

ABSTRACT

Compositions that are used in nucleic acid amplification in vitro are disclosed, which include a target specific universal (TSU) promoter primer or promoter provider oligonucleotide that includes a target specific (TS) sequence that hybridizes specifically to a target sequence that is amplified and a universal (U) sequence that is introduced into the sequence that is amplified, by using a primer for the universal sequence. Methods of nucleic acid amplification in vitro are disclosed that use one or more TSU oligonucleotides to attached a U sequence to a target nucleic acid in a target capture step and then use a primer for a U sequence in subsequent amplification steps performed in substantially isothermal conditions to make amplification products that contain a U sequence that indicates the presence of the target nucleic acid in a sample.

RELATED APPLICATIONS

This application is a continuation under 35 U.S.C. 120 of U.S. patentapplication Ser. No. 14/109,709, filed Dec. 17, 2013, which is acontinuation under 35 U.S.C. 120 of U.S. patent application Ser. No.13/460,341, filed Apr. 30, 2012, issued as U.S. Pat. No. 8,642,268,which is a continuation under 35 U.S.C. 120 of U.S. patent applicationSer. No. 11/962,072, filed Dec. 20, 2007, issued as U.S. Pat. No.8,198,027, which claims the benefit under 35 U.S.C. 119(e) of U.S.Provisional Patent Application No. 60/871,451, filed Dec. 21, 2006, eachof which is incorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII Copy is namedGP198-05-CN3_ST25.txt and is 18 kilo-bytes in size.

FIELD

This invention relates to molecular biology, more specifically to invitro amplification of nucleic acids which is useful for increasing thenumber of copies of a nucleic acid sequence to provide sufficient copiesto be readily detected.

BACKGROUND

Nucleic acid amplification provides a means for making more copies of anucleic acid sequence that is relatively rare or unknown, foridentifying the source of nucleic acids, or for making sufficientnucleic acid to provide a readily detectable amount. Amplification isuseful in many applications, for example, in diagnostics, drugdevelopment, forensic investigations, environmental analysis, and foodtesting.

Many methods for amplifying nucleic acid sequences in vitro are known,including polymerase chain reaction (PCR), ligase chain reaction (LCR),replicase-mediated amplification, strand-displacement amplification(SDA), “rolling circle” types of amplification, and varioustranscription associated amplification methods. These known methods usedifferent techniques to make amplified sequences, which usually aredetected by using a variety of methods. PCR amplification uses a DNApolymerase, oligonucleotide primers, and thermal cycling to synthesizemultiple copies of both strands of a double-stranded DNA (dsDNA) ordsDNA made from a cDNA (U.S. Pat. Nos. 4,683,195, 4,683,202, and4,800,159, Mullis et al.). LCR amplification uses an excess of twocomplementary pairs of single-stranded probes that hybridize tocontiguous target sequences and are ligated to form fused probescomplementary to the original target, which allows the fused probes toserve as a template for further fusions in multiple cycles ofhybridization, ligation, and denaturation (U.S. Pat. No. 5,516,663 andEP 0320308 B1, Backman et al.). Replicase-mediated amplification uses aself-replicating RNA sequence attached to the analyte sequence and areplicase, such as Qβ-replicase, to synthesize copies of theself-replicating sequence specific for the chosen replicase, such as aQβ viral sequence (U.S. Pat. No. 4,786,600, Kramer et al.). Theamplified sequence is detected as a substitute or reporter molecule forthe analyte sequence. SDA uses a primer that contains a recognition sitefor a restriction endonuclease which allows the endonuclease to nick onestrand of a hemimodified dsDNA that includes the target sequence,followed by a series of primer extension and strand displacement steps(U.S. Pat. No. 5,422,252A, Walker et al., and U.S. Pat. No. 5,547,861,Nadeau et al.). Rolling circle types of amplification rely on a circularor concatenated nucleic acid structure that serves as a template used toenzymatically replicate multiple single-stranded copies from thetemplate (e.g., U.S. Pat. No. 5,714,320, Kool, and U.S. Pat. No.5,834,252, Stemmer et al.). Transcription associated amplificationrefers to methods that amplify a sequence by producing multipletranscripts from a nucleic acid template. Such methods generally use oneor more oligonucleotides, of which one provides a promoter sequence, andenzymes with RNA polymerase and DNA polymerase activities to make afunctional promoter sequence near the target sequence and thentranscribe the target sequence from the promoter (e.g., U.S. Pat. Nos.5,399,491 and 5,554,516, Kacian et al., U.S. Pat. No. 5,437,990, Burg etal., WO 1988010315 A1, Gingeras et al., U.S. Pat. No. 5,130,238, Maleket al., U.S. Pat. Nos. 4,868,105 and 5,124,246, Urdea et al., and US2006-0046265 A1, Becker et al.). Nucleic acid amplification methods mayamplify a specific target sequence (e.g., a gene sequence), a group ofrelated target sequences, or a surrogate sequence, which may be referredto as a tag or reporter sequence that is amplified and detected in placeof the analyte sequence. The surrogate sequence is only amplified if theanalyte target sequence is present at some point during the reaction.

Modified nucleic acid amplification methods may amplify more than onepotential target sequence by using “universal” primer(s) or universalpriming. One form of PCR amplification uses universal primers that bindto conserved sequences to amplify related sequences in a PCR reaction(Okamoto et al., 1992, J. Gen. Virol. 73(Pt. 3):673-9, Persing et al,1992, J. Clin. Microbiol. 30(8):2097-103). Methods that use universalprimers often are paired with use of a species-specific, gene-specificor type-specific primer or primers to generate an amplified sequencethat is unique to a species, genetic variant, or viral type, which maybe identified by sequencing or detecting some other characteristic ofthe amplified nucleic acid. For example, a method may use one universalprimer and one specific primer in the same amplification step. Foranother example, a method may use “nested” PCR in which a pair ofuniversal primers are used in an initial amplification step to amplifymany potential target sequences, followed by use of a pair of specificprimers in subsequent amplification steps to amplify one or morespecific target sequences contained in the initial amplicons.

Anchored PCR is another modified PCR method that uses a universal primeror an “adapter” primer to amplify a sequence which is only partiallyknown. Anchored PCR introduces an “adaptor” or “universal” sequence intoa cDNA and then uses a primer that binds to the introduced sequence insubsequent amplification steps. Generally, anchored-PCR uses a primerdirected to a known sequence to make a cDNA, adds a known sequence(e.g., poly-G) to the cDNA or uses a common sequence in the cDNA (e.g.,poly-T), and performs PCR by using a universal primer that binds to theadded or common sequence in the cDNA and a downstream target-specificprimer (Loh et al., 1989, Science 243(4888):217-20; Lin et al., 1990,Mol. Cell. Biol. 10(4):1818-21). Nested PCR may use primer(s) thatcontain a universal sequence unrelated to the analyte target sequence toamplify nucleic acid from unknown target sequences in a reaction(Sullivan et al, 1991, Electrophoresis 12(1):17-21; Sugimoto et al.,1991, Agric. Biol. Chem. 55(11):2687-92).

Other forms of amplification use a probe or probe set to introduceuniversal priming sites located upstream and downstream of atarget-specific sequence and adapter sequence(s), which may be referredto as molecular zip-codes. The upstream and downstream priming sites areused to amplify a nucleic acid that contains the adapter sequence(s)which are detected, usually on an array, to identify the target presentin the reaction (U.S. Pat. Nos. 6,812,005 and 6,890,741, Fan et al.).The two probes that bind in close proximity on a target sequence may beligated together before being amplified by using the upstream anddownstream universal priming sites.

Alternative assay methods may use probe hybridization and linear signalamplification by using a common sequence that is included in a varietyof analyte-specific probes (e.g., US 20070111200, Hudson et al.). Thismethod uses a labeled cassette that contains a sequence complementary tothe common sequence to detect multiple analytes.

SUMMARY

A composition is disclosed that includes a TSU promoter oligonucleotidethat includes a 5′ promoter sequence, an internal first universalsequence (U1), and a 3′ first target specific sequence (TS1) that bindsspecifically to a target sequence contained in a target nucleic acid,wherein the TSU promoter oligonucleotide is a TSU promoter primer thathas a 3′ terminus that is capable of being extended by a polymerase, oris a TSU promoter provider oligonucleotide that has a blocked 3′terminus that is incapable of being extended by a polymerase, a TSUnon-promoter primer oligonucleotide made up of a 5′ second universalsequence (U2) and a 3′ second target specific sequence (TS2) which isdifferent from the TS1, and a means for directly or indirectly joiningthe TSU promoter oligonucleotide to the TSU non-promoter primeroligonucleotide, thereby forming a target specific universal (TSU)primer complex. In one embodiment, the means for directly joining theTSU promoter oligonucleotide to the TSU non-promoter primeroligonucleotide is a covalent linkage. In another embodiment, thecovalent linkage is formed via a polynucleotide linker sequence, whichmay be a covalent linkage formed via a non-nucleotide abasic linkercompound. Another embodiment uses a means for indirectly joining the TSUpromoter oligonucleotide to the TSU non-promoter primer oligonucleotidethat is a non-covalent linkage of members of a binding pair to join theTSU promoter oligonucleotide and the TSU non-promoter primeroligonucleotide to a support, in which one member of the binding pair ispresent on the TSU promoter oligonucleotide or the TSU non-promoterprimer oligonucleotide and the other member of the binding pair isattached to the support. In another embodiment, the means for directlyjoining the TSU promoter oligonucleotide to the TSU non-promoter primeroligonucleotide is a hybridization complex between a first sequence onthe TSU promoter oligonucleotide and a second sequence on the TSUnon-promoter primer that is complementary to the first sequence on theTSU promoter oligonucleotide. The means for indirectly joining the TSUpromoter oligonucleotide to the TSU non-promoter primer oligonucleotidemay be a hybridization complex that includes an S-oligonucleotide thatcontains a first sequence complementary to a sequence in the TSUpromoter oligonucleotide and a second sequence complementary to asequence in the TSU non-promoter primer oligonucleotide. In oneembodiment the S-oligonucleotide contains a first sequence complementaryto the universal sequence in the TSU promoter oligonucleotide and theS-oligonucleotide contains a second sequence complementary to theuniversal sequence in the TSU non-promoter primer oligonucleotide. Thecomposition may also include a target specific capture oligonucleotidethat contains a sequence that hybridizes specifically to a sequence inthe target nucleic acid of the TSU promoter oligonucleotide and the TSUnon-promoter primer at a sequence that is different from the sequence inthe target nucleic acid that hybridizes to the TS sequence of the TSUpromoter oligonucleotide or the TS sequence of the TSU non-promoterprimer, and contains a means for binding the target nucleic acid to asupport. The composition may also include a universal promoter primermade up a 5′ promoter sequence and a 3′ universal sequence that is thesame as the universal sequence of the TSU promoter oligonucleotide.Another embodiment is a composition that further includes a universalprimer made up a universal sequence that is the same as the universalsequence of the TSU non-promoter primer oligonucleotide. The compositionmay also include a blocker oligonucleotide that hybridizes specificallyto a sequence in a target nucleic acid strand that is different than thesequence that the TS sequence of the TSU promoter oligonucleotide or theTS sequence of the TSU non-promoter primer oligonucleotide binds to inthe target nucleic acid strand, wherein the blocker oligonucleotide hasa 3′ blocked terminus that is not capable of being extended by apolymerase. In some embodiments that include an S-oligonucleotide, it ismade up of (1) a first terminal region sequence that is complementary tothe U1 sequence of the TSU promoter primer and (2) a second terminalregion sequence that is complementary to the U2 sequence of the TSUnon-promoter primer, and (3) a linking moiety that links the first andsecond terminal region sequences. The linking moiety may be anon-nucleic acid chemical compound that covalently links the first andsecond terminal region sequences. The composition may also include atleast one universal promoter primer made up of a 5′ promoter sequenceand a 3′ U1 sequence and at least one target specific primer (TSP) madeup of a sequence that is complementary to a sequence contained in an RNAtranscript made from a double stranded DNA that contains a cDNA sequencemade from synthetic extension of the 3′ end of the TSU promoter primeroligonucleotide.

Also disclosed is a method of amplifying a target nucleic acidcomprising the steps of: isolating a target nucleic acid from a mixtureby binding to the target nucleic acid a target capture probe that bindsspecifically to the target nucleic acid and provides a means forattaching the bound target nucleic acid to a support that is separatedfrom the mixture and further hybridizing to the target nucleic acid inthe mixture a target specific universal (TSU) primer complex made up of(1) a TSU promoter primer oligonucleotide that includes a 5′ promotersequence, an internal first universal sequence (U1), and a 3′ firsttarget specific sequence (TS1) that binds specifically to a targetsequence contained in a target nucleic acid, and a 3′ terminus that iscapable of being extended by a polymerase, (2) a TSU non-promoter primeroligonucleotide made up of a 5′ second universal sequence (U2) and a 3′second target specific sequence (TS2) which is different from the TS1,and (3) a means for directly or indirectly joining the TSU promoteroligonucleotide to the TSU non-promoter primer oligonucleotide. Themethod includes hybridizing the TSU promoter primer oligonucleotide to atarget sequence in the target nucleic acid via a TS sequence in the TSUpromoter primer, synthetically extending the 3′ terminus of the TSUpromoter primer oligonucleotide hybridized to the target nucleic acid byusing a polymerase in vitro nucleic acid synthesis in which the targetnucleic acid is a template to make a first cDNA strand, hybridizing theTSU non-promoter primer oligonucleotide to the first cDNA strand byspecific hybridization of the TS sequence in the TSU non-promoter primeroligonucleotide to a target sequence contained in the first cDNA strand,synthetically extending the 3′ terminus of the TSU non-promoter primeroligonucleotide hybridized to the first cDNA strand by a polymerase invitro nucleic acid synthesis to made a second DNA strand, thereby makinga substantially double-stranded DNA that contains a functional promotersequence and the U1 sequence, enzymatically transcribing RNA transcriptsfrom the functional promoter sequence of the substantiallydouble-stranded DNA to make RNA transcripts that contain a 5′ U1 regionsequence, a first target specific sequence (TS1), a second targetspecific sequence (TS2′), and a 3′ universal sequence (U2′) that iscomplementary to the U2 sequence, hybridizing a universal primeroligonucleotide (UP2) that contains a universal sequence U2 to the RNAtranscript at the U2′ sequence, under isothermal conditions,synthetically extending the 3′ terminus of the UP2 by enzymatic in vitronucleic acid synthesis to made a cDNA strand, and enzymatically removingthe RNA transcript strand, hybridizing a universal promoter primeroligonucleotide (UP1) that contains a universal sequence U1 to the cDNAmade in the previous step at the U1′ sequence, under isothermalconditions, synthetically extending the 3′ terminus of the UP1 byenzymatic in vitro nucleic acid synthesis to made a dsDNA that containsa functional promoter, and transcribing multiple RNA transcripts fromthe functional promoter of the dsDNA, which transcripts areamplification products that may serve as templates for further enzymaticin vitro nucleic acid synthesis under isothermal conditions by bindingthe UP2 primer and repeating the synthetic steps. The method may alsoinclude the step of detecting the amplification products to indicate thepresence of an analyte in the mixture from which the target nucleic acidwas isolated.

Another disclosed method of amplifying a target nucleic acid includesisolating a target nucleic acid from a mixture by binding to the targetnucleic acid a target capture probe that binds specifically to thetarget nucleic acid and provides a means for attaching the bound targetnucleic acid to a support that is separated from the mixture and furtherhybridizing to the target nucleic acid in the mixture a target specificuniversal (TSU) primer complex made up of (1) a TSU promoteroligonucleotide that includes a 5′ promoter sequence, an internal firstuniversal sequence (U1), and a 3′ first target specific sequence (TS1)that binds specifically to a target sequence contained in a targetnucleic acid, wherein the TSU promoter oligonucleotide is a TSU promoterprovider oligonucleotide that has a blocked 3′ terminus that isincapable of being extended by a polymerase, (2) a TSU non-promoterprimer oligonucleotide made up of a 5′ second universal sequence (U2)and a 3′ second target specific sequence (TS2) which is different fromthe TS1, and (3) a means for directly or indirectly joining the TSUpromoter oligonucleotide to the TSU non-promoter primer oligonucleotide.The method steps also include hybridizing the TSU non-promoter primeroligonucleotide to a target sequence in the target nucleic acid via theTS sequence in the TSU non-promoter primer, optionally hybridizing ablocker oligonucleotide with a 3′ blocked end that is incapable of beingextended synthetically by a polymerase to a sequence on the targetnucleic acid that is downstream from the position that the TSUnon-promoter primer oligonucleotide hybridizes in the target nucleicacid, synthetically extending the 3′ terminus of the TSU non-promoterprimer hybridized to the target nucleic acid by using a polymerase invitro nucleic acid synthesis in which the target nucleic acid is atemplate to make a first cDNA strand, hybridizing the TSU promoterprovider oligonucleotide to the first cDNA strand by specifichybridization of the TS sequence in the TSU promoter provideroligonucleotide to a target sequence contained in the first cDNA strand,synthetically extending the 3′ terminus of the first cDNA by usingsequence in the TSU promoter provider as a template to make asubstantially double-stranded DNA that contains a functional promotersequence and the U1 sequence, enzymatically transcribing RNA transcriptsfrom the functional promoter sequence to make RNA transcripts thatcontain a 5′ U1 region sequence, a first target specific sequence (TS1),a second target specific sequence (TS2′), and a 3′ universal sequence(U2′) that is complementary to the U2 sequence, hybridizing a universalprimer oligonucleotide (UP2) that contains a universal sequence U2 tothe RNA transcript at the U2′ sequence, under isothermal conditions,synthetically extending the 3′ terminus of the UP2 by enzymatic in vitronucleic acid synthesis to made a cDNA strand, and enzymatically removingthe RNA transcript strand, hybridizing a universal promoteroligonucleotide (UP1) that contains a promoter sequence, a universalsequence U1, and a 3′ blocked end to the cDNA made in the previous stepat the U1′ sequence, under isothermal conditions, syntheticallyextending the 3′ terminus of the cDNA to make a functionaldouble-stranded promoter by using the UP1 oligonucleotide as a templateand by enzymatic in vitro nucleic acid synthesis to made a dsDNA thatcontains a functional promoter, and transcribing multiple RNAtranscripts from the functional promoter of the dsDNA, which transcriptsare amplification products that may serve as templates for furtherenzymatic in vitro nucleic acid synthesis under isothermal conditions bybinding the UP2 primer and repeating the synthetic steps. The method mayfurther include the step of detecting the amplification products toindicate the presence of an analyte in the sample from which the targetnucleic acid was isolated.

Also disclosed is a method of amplifying a target nucleic acid thatincludes steps of isolating a target nucleic acid from a mixture bybinding to the target nucleic acid a target capture probe that bindsspecifically to the target nucleic acid and provides a means forattaching the bound target nucleic acid to a support that is separatedfrom the mixture and further hybridizing to the target nucleic acid inthe mixture a target specific universal (TSU) promoter primeroligonucleotide that includes a 5′ promoter sequence, an internal firstuniversal sequence (U1), and a 3′ first target specific sequence (TS1)that binds specifically to a target sequence contained in a targetnucleic acid, and a 3′ terminus that is capable of being extended by apolymerase, synthetically extending the 3′ terminus of the TSU promoterprimer oligonucleotide hybridized to the target nucleic acid by using apolymerase in vitro nucleic acid synthesis in which the target nucleicacid is a template to make a first cDNA strand, adding to theamplification reaction mixture a target specific (TS) non-promoterprimer that contains a second target specific sequence (TS2) which isdifferent from the TS1, hybridizing the TS non-promoter primeroligonucleotide to the first cDNA strand by specific hybridization ofthe TS2 sequence to a target sequence contained in the first cDNAstrand, synthetically extending the 3′ terminus of the TS non-promoterprimer oligonucleotide hybridized to the first cDNA strand by apolymerase in vitro nucleic acid synthesis to made a second DNA strand,thereby making a substantially double-stranded DNA that contains afunctional promoter sequence and the U1 sequence, enzymaticallytranscribing RNA transcripts from the functional promoter sequence ofthe substantially double-stranded DNA to make RNA transcripts thatcontain a 5′ U1 region sequence, a first target specific sequence (TS1),a second target specific sequence (TS2′), hybridizing a universalpromoter primer oligonucleotide that contains a universal sequence U1′to the RNA transcript at the U1 sequence, under isothermal conditions,synthetically extending the 3′ terminus of the universal promoter primerby enzymatic in vitro nucleic acid synthesis to made a cDNA strand, andenzymatically removing the RNA transcript strand, hybridizing a TSnon-promoter primer oligonucleotide to a specific sequence in the cDNAmade in the previous step, under isothermal conditions, syntheticallyextending the 3′ terminus of the TS non-promoter primer by enzymatic invitro nucleic acid synthesis to made a dsDNA that contains a functionalpromoter, and transcribing multiple RNA transcripts from the functionalpromoter of the dsDNA, which transcripts are amplification products thatmay serve as templates for further enzymatic in vitro nucleic acidsynthesis under isothermal conditions by repeating the synthetic steps.The method may further include detecting the amplification products toindicate the presence of an analyte in the mixture from which the targetnucleic acid was isolated.

Another disclosed method of amplifying a target nucleic acid includesthe steps of isolating a target nucleic acid from a mixture by bindingto the target nucleic acid a target capture probe that bindsspecifically to the target nucleic acid and provides a means forattaching the bound target nucleic acid to a support that is separatedfrom the mixture and further hybridizing to the target nucleic acid inthe mixture a TSU non-promoter primer oligonucleotide made up of a 5′universal sequence (U2) and a 3′ target specific sequence (TS2),hybridizing the TSU non-promoter primer oligonucleotide to a targetsequence in the target nucleic acid via the TS2 sequence to acomplementary sequence in the target nucleic acid, hybridizing a blockeroligonucleotide with a 3′ blocked end that is incapable of beingextended synthetically by a polymerase to a sequence on the targetnucleic acid that is downstream from the position that the TSUnon-promoter primer oligonucleotide hybridizes in the target nucleicacid, synthetically extending the 3′ terminus of the TSU non-promoterprimer hybridized to the target nucleic acid by using a polymerase invitro nucleic acid synthesis in which the target nucleic acid is atemplate to make a first cDNA strand, hybridizing to the first cDNAstrand a target specific TS promoter provider oligonucleotide thatincludes a 5′ promoter sequence and a 3′ target specific sequence (TS1)that binds specifically to a target sequence contained in a targetnucleic acid, and a blocked 3′ terminus that is incapable of beingextended by a polymerase, by specific hybridization of the TS1 sequenceto a complementary sequence in the first cDNA strand, syntheticallyextending the 3′ terminus of the first cDNA by using sequence in the TSpromoter provider as a template to make a substantially double-strandedDNA that contains a functional promoter sequence and a TS1 sequence,enzymatically transcribing RNA transcripts from the functional promotersequence to make RNA transcripts that contain a 5′ target specificsequence TS1, a target specific sequence TS2′ and a U2′ sequence,hybridizing a universal primer oligonucleotide (UP2) that contains auniversal sequence U2 to the RNA transcript at the U2′ sequence, underisothermal conditions, synthetically extending the 3′ terminus of theUP2 by enzymatic in vitro nucleic acid synthesis to made a cDNA strand,and enzymatically removing the RNA transcript strand, hybridizing a TSpromoter provider oligonucleotide that contains a promoter sequence anda 3′ blocked end to the cDNA made in the previous step, under isothermalconditions, synthetically extending the 3′ terminus of the cDNA to makea functional double-stranded promoter by using the TS promoter provideroligonucleotide as a template and by enzymatic in vitro nucleic acidsynthesis to made a dsDNA that contains a functional promoter, andtranscribing multiple RNA transcripts from the functional promoter ofthe dsDNA, which transcripts are amplification products that may serveas templates for further enzymatic in vitro nucleic acid synthesis underisothermal conditions by repeating the synthetic steps. The method mayalso include detecting the amplification products to indicate thepresence of an analyte in the sample from which the target nucleic acidwas isolated.

The accompanying drawings, which constitute a part of the specification,illustrate some embodiments of the invention. These drawings, togetherwith the description, serve to explain and illustrate the principles ofthe invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic drawing showing: a three-component target-specificuniversal (TSU) primer complex that includes a TSU promoter primer madeup of a 5′ promoter sequence (solid line) labeled P, a universalsequence (dashed line) labeled U1, and a 3′ target-specific sequence(double lines) labeled TS1, which is hybridized to an S-oligonucleotide(S-shaped dotted line) that includes a 5′ universal sequence labeled U1′and a 3′ universal sequence labeled U2′, which is hybridized to a TSUnon-promoter primer made up of a 5′ universal sequence (dashed line)labeled U2 and a 3′ target-specific sequence (double line) labeled TS2;a target-specific capture oligonucleotide made up of a 5′target-specific sequence (double line) labeled TS3 and a 3′ binding pairmember (triple line) labeled BPM; a universal promoter primer (UP1) madeup of a 5′ promoter sequence (solid line) labeled P and a 3′ universalsequence (dashed line) labeled U1; and a universal non-promoter primer(UP2) made up of a universal sequence (dashed line) labeled U2.

FIG. 2 is a schematic drawing illustrating target capture in which: (1)target capture reagent (TCR) contains multiple three-componenttarget-specific universal (TSU) primer complexes (see FIG. 1) specificfor three different targets (labeled TSUa, TSUb, TSUc) and captureprobes specific for the three different targets in which the BPM isshown as poly-A sequences (AAA) and the target-specific sequences arelabeled TSa, TSb, and TSc; (2) TCR is mixed with a sample that contains“Target a”, which allows the TSUa primer complex to hybridize to Targeta and the TSa capture probe to hybridize to Target a; (3) the poly-Asequence of the TSa capture probe hybridizes to an immobilized probe(poly-T sequence shown as TTTT) which is attached to a support (shadedcircle), which allows the complex attached to the support to beseparated from the mixture to retrieve the captured target and TSUprimer complex; and (4) the portion containing the unbound TSU primercomplexes (labeled TSUb and TSUb) is discarded as waste.

FIG. 3 is a schematic drawing that illustrates a three-component TSUprimer complex which is attached to a target strand via hybridization ofthe TS1 sequence of the TSU promoter primer to a complementary TS1′sequence in the target nucleic acid, which is attached to a support(shaded circle) via hybridization of the target specific TS3 sequence ofa capture probe to a complementary TS3′ sequence of the target nucleicacid and the poly-A portion of the capture probe is hybridized to animmobilized poly-T probe that is attached to the support. Verticalconnecting lines (|||||) indicate sequence hybridization. The TSU primercomplex is made up of the TSU non-promoter primer hybridized at its U2sequence region to the complementary U2′ sequence region of theS-oligonucleotide which has a 3′ blocked end (

) and a 5′ region that is hybridized at its U1′ sequence region to acomplementary U1 sequence region in the TSU promoter primer thatincludes a 5′ promoter sequence region (solid line P) and a 3′ targetspecific sequence region (TS1) which is complementary to the TS1′sequence in the target strand. The target strand also contains anothertarget specific sequence region (TS2) which is the same as the TS2region of the TSU non-promoter primer. The capture probe contains a 5′target specific sequence (TS3) that is complementary to part of thetarget strand (sequence TS3′) and a 3′ poly-A sequence that iscomplementary to a poly-T sequence that serves as the BPM of theimmobilized probe.

FIG. 4 is a schematic drawing that illustrates a TSU primer complex inwhich the upper strand is a TSU non-promoter primer made up of a 3′target specific region (TS2) and a 5′ universal sequence region, labeledU2(+), which is hybridized to a complementary 3′ U2′ sequence region ofthe S-oligonucleotide (labeled S-oligo) which is contains an abasicspacer that links the 3′ U2′ sequence to a 5′ U1′ sequence region thatis the complement of and hybridized to the U1(−) sequence region in theTSU promoter primer that includes a 5′ promoter sequence (P) and a 3′target specific sequence region (TS1). The illustrated S-oligonucleotideincludes a 3′ blocked end in which terminal bases are joined by a 3′ to3′ linkage (labeled 3′-3′C) and an internal abasic compound (e.g., (C9)₂or (C9)₃) that is a spacer that covalently joins the 5′ U1′ sequence andthe 3′ U2′ sequence.

FIG. 5 is a schematic drawing that illustrates the product that resultsfrom an initial synthetic step of the initial amplification phase inwhich the 3′ end of the TSU promoter primer, hybridized via its TS1sequence to the complementary TS1′ sequence in an RNA template strand(narrow solid line), has been synthetically extended to make a firststrand cDNA (wider solid line) by using a reverse transcriptase (RT)polymerase. The RNA template strand also contains a TS2 sequence that iscomplementary to the TS2′ sequence made in the first strand cDNA.

FIG. 6 is a schematic drawing that illustrates the first strand cDNAproduct (as shown in FIG. 5) following degradation of the RNA templatestrand that was shown in FIG. 5, in which the cDNA contains a 5′promoter sequence (P), a universal sequence (U1), a target-specificsequence (TS1), a cDNA sequence that was made from the template strandand that contains a second target-specific sequence (TS2′).

FIG. 7 is a schematic drawing that illustrates the product that resultsfrom a second synthetic step in the initial phase of amplification. Thisproduct results from hybridization of the TSU non-promoter primer to thefirst strand cDNA product (see FIG. 6) by hybridizing the TS2 sequenceof the TSU non-promoter primer to the complementary TS2′ sequence of thecDNA and extending the 3′ end of the TSU non-promoter primer by using aDNA polymerase (shaded rectangle) to make a complementary second strandof DNA. The second strand contains the primer's 5′ U2 sequence and TS2sequence, the complementary sequence to the first strand cDNA whichincludes a target specific sequence TS1′, a universal sequence U1′ and a3′ sequence that is complementary to the promoter sequence of the cDNA,thus making a double-stranded DNA that contains a functional promotersequence.

FIG. 8 is a schematic drawing that illustrates the substantially dsDNAmade up of the first strand cDNA and the second strand DNA (see FIG. 7)and three RNA transcripts (broader lines) above the dsDNA. RNAtranscripts are made by transcription that initiates at the functionaldouble-stranded promoter sequence (P) by using its respective RNApolymerase (shaded area labeled RNA Pol). RNA transcripts include, in a5′ to 3′ direction, a 5′ U1 sequence, a TS1 sequence, a transcript fromthe target strand, a TS2′ sequence, and a 3′ U2′ sequence.

FIG. 9 is a schematic drawing showing a single RNA transcript, asillustrated in FIG. 8, from the first phase of isothermal amplificationwith terminal universal sequences, U1 and U2′, which flank the targetspecific sequences TS1 and TS2′, which flank the transcript of othertarget strand sequence, and a universal primer (UP2) that includessequence U2 that is complementary to sequence U2′ in the transcript.

FIG. 10 is a schematic drawing showing the steps in the second phase ofisothermal amplification in which RNA transcripts (as illustrated inFIG. 9) enter the system at the lower left where the RNA transcripthybridizes to the universal primer UP2 via complementary pairing of theU2′ and U2 sequences (hybridization shown by vertical lines |||||) andreverse transcriptase enzyme (open circle labeled RT) attaches to UP2and uses its RNA directed DNA polymerase activity to enzymaticallyextend the UP2 primer by using the RNA transcript as a template. Thenext step, after the arrow pointing to the right, shows the resultingcDNA (lower strand) hybridized to the RNA template (upper strand), whichafter the upward pointing arrow, is digested by RNAse H activity of theRT enzyme that leaves the cDNA strand. After the next upward pointingarrow, the cDNA is hybridized via its U1′ sequence to the complementaryU1 sequence of the universal promoter primer (UP1) which includes a 5′promoter sequence (P) and the UP1 primer is extended by DNA directed DNApolymerase activity of the RT enzyme to make a dsDNA that is illustratedat the top of the circle, above the arrow pointing upward and leftward.The dsDNA contains two universal sequences per strand (U1 and U2′ on theupper strand and U1′ and U2 on the lower strand) which flank targetspecific sequences (TS1, TS2′ and the intervening sequence on the upperstrand and TS1′ and TS2 and the intervening sequence on the lowerstrand), and a functional promoter (P). Following the arrow downward tothe left, the functional promoter interacts with a RNA polymerase (ovallabeled RNA Pol) specific for the promoter sequence to make transcriptsfrom the dsDNA, which are shown after the next downward pointing arrow,to result in 100 to 1000 transcripts or RNA amplicons which contain twouniversal sequences (U1 and U2′) and target specific sequences (TS1 andTS2′ and the intervening sequence). Following the next arrow downwardand to the right, these RNA transcripts enter the amplification systemand are used as templates for further isothermal amplification in acyclic manner as shown, repeating the steps as described above for thefirst phase RNA transcripts.

FIG. 11 is a schematic drawing of two embodiments of TSU primers that donot include an S-oligonucleotide but which may be used in the firstphase of isothermal amplification which is performed using TSU primersattached to a support, followed by the second phase of isothermalamplification performed in solution phase by using the universal primers(UP1 and UP2). In Embodiment 1, a TSU non-promoter primer and a TSUpromoter primer are linked together, covalently or non-covalently, andattached to a support via a first binding pair member (shaded arrowlabeled BPM1) which binds specifically to a second binding pair member(dark chevron labeled BPM2) attached to the support (shaded rectangle).In Embodiment 2, the TSU non-promoter primer and TSU promoter primer areseparate oligonucleotides which are separately attached to the samesupport via a BPM1 attached to each oligomer, which binds specificallyto a separate binding pair member, BPM2, attached to the support (shadedcircle). For both Embodiment 1 and 2, universal primers (UP1 and UP2)are provided in solution phase and are unattached to a support.

FIG. 12 is a schematic drawing showing structures used in a targetcapture (TC) step with initial primer attachment (left side, labeled A.)and primers used in the second phase of isothermal amplification (rightside, labeled B.), for Embodiment 1 (upper half above the line) andEmbodiment 2 (lower half below the line). In Embodiment 1, the TC step(left side, upper half) includes a capture complex made up of the targetnucleic acid attached to a support, via a target specific capture probethat hybridizes to the target strand (shown by vertical lines between ashort horizontal line and the longer horizontal line representing thetarget strand) and also hybridizes via a poly-A sequence to animmobilize poly-T sequence attached to the support (shaded circle). Thetarget nucleic acid is attached at another location to a TSU primercomplex that includes the TSU promoter primer hybridized specifically toa sequence in the target strand and to an S-oligonucleotide that ishybridized to a TSU non-promoter primer (substantially as shown in FIG.3). In Embodiment 1, the second phase of amplification (right side,upper half) uses two universal primers: a universal promoter primer(UP1) and a universal non-promoter primer (UP2) which hybridizes to acomplementary sequence introduced in the RNA transcript by use of theTSU primer complex. In Embodiment 2, the TC step (left side, lower half)includes the capture complex as shown for Embodiment 1 and only the TSUpromoter primer hybridized via a target-specific sequence at anotherlocation on the target strand, and the second phase of amplification(right side, lower half) uses one universal promoter primer (UP1) andone target specific primer (TSP).

FIG. 13 is a schematic drawing showing the steps in the second phase ofisothermal amplification substantially as shown in FIG. 10, except thatRNA transcripts from the first and/or second phases (lower left) arehybridized to a target specific primer (TSP) that is extended by RT tosynthesize the cDNA strand (lower right) using the RNA transcripts astemplates, and no U2 or U2′ universal sequences are present.

FIG. 14 is a schematic drawing showing an embodiment in which (lowerleft) a TSU promoter primer used in a first phase of amplification isattached to a support via a first binding pair member (BPM1) that bindsspecifically to a second binding pair member (BPM2) attached to thesupport (shaded circle), and a mixture of universal promoter primers(UP1) and target specific primers (TSP) in solution phase are used inthe second phase of amplification.

FIG. 15 is a schematic drawing showing components in an embodiment inwhich the top portion of the diagram shows a hybridization complex madein the Target Capture step, made up of the Target nucleic acid strandhybridized to a target capture (TC) probe that has an unbound poly-Atail and a TS sequence hybridized to a 5′ portion of the target strand,a Blocker oligonucleotide hybridized to the target strand downstreamfrom the position hybridized to the TC probe, and a TSU primerhybridized to a 3′ portion of the target strand via a TS sequence withan unhybridized universal (U) sequence; and the lower portion of thediagram shows that the nucleic acids present in single-primer isothermalamplification which include (1) the target amplicon consisting of a 5′ Usequence, an internal TS sequence, and a 3′ sequence copied from thetarget strand by extension of the TSU primer, (2) a TS promoter providerthat includes a 5′ promoter (P) sequence, a 3′ TS sequence, and ablocked 3′ end ({circle around (×)}), and (3) a universal primerconsisting of a universal sequence (U′) complementary to the universalsequence of the target amplicon.

FIG. 16 is a schematic drawing showing components in an embodiment inwhich the top portion of the diagram shows a hybridization complex madein the Target Capture step, made up of the Target nucleic acid strandhybridized to a target capture (TC) probe that has an unbound poly-Atail and a TS sequence hybridized to a 5′ portion of the target strand,a Blocker oligonucleotide hybridized to the target strand downstreamfrom the position hybridized to the TC probe, and a TSU primer complexmade up of (top strand) a TSU promoter provider with a 3′ blocked end({circle around (×)}), an S-oligomer (middle strand, substantially as inFIG. 3), and a TSU primer (lower strand) hybridized to a 3′ portion ofthe target strand via a TS sequence with its universal (U2) sequencehybridized to a complementary (U2′) sequence in the S-oligomer; and thelower portion of the diagram shows that nucleic acids present insingle-primer isothermal amplification which include (1) the TSUpromoter provider hybridized via its TS1 sequence to the extensionproduct made by extension of the TS2 sequence of the TSU primer whichincludes its U2 universal sequence, (2) a promoter provideroligonucleotide that includes a 5′ promoter (P) sequence, a 3′ U1′universal sequence, and a blocked 3′ end ({circle around (×)}), and (3)a universal primer consisting of a universal sequence (U2′)complementary to the U2 universal sequence.

FIG. 17 is a schematic drawing of an embodiment showing two TSUoligonucleotides in a hybridization complex that is hybridized to atarget strand via the TS1 sequence of a TSU primer which also includes aU1 sequence and a promoter complementary sequence (P′), which ishybridized to a TSU promoter provider oligonucleotide via hybridizationof the complementary P′ sequence and the P sequence of the TSU promoterprovider oligonucleotide which also contains a U2 sequence, a TS2sequence and a blocked 3′ end (

).

FIG. 18 is a schematic drawing of an embodiment showing two TSUoligonucleotides joined covalently via a non-nucleotide linker(—C₉—C₉—). This forms a complex made up of a TSU promoter provider thatincludes a blocked 3′ end, and TS2, U2 and promoter (P) sequences in a3′ to 5′ orientation linked to a TSU primer that includes U1 and TS1sequences in a 5′ to 3′ orientation, providing one extendable 3′terminus in the complex which is hybridized to a target strand via theTS1 sequence of the TSU primer. Also shown hybridized to the Targetstrand are a blocker oligonucleotide and a TC probe, hybridized to thetarget via its TS sequence and shown with an un-hybridized tailsequence.

FIG. 19 shows data obtained from an isothermal amplification of a singletarget (“PCA3 uniplex” panel) present in samples at 10², 10⁴ and 10⁶copies per reaction, and of two targets (“PCA3/PSA duplex (oligos)”panel) present in samples at 10⁶ copies per reaction, in whichamplification products were detected in real time by using afluorescent-labeled probe. For both panels, the x-axis shows cycles ofamplification and the y-axis shows fluorescence units.

DETAILED DESCRIPTION

The invention encompasses compositions that include one or moretarget-specific universal (TSU) oligonucleotide primers that includeboth a target-specific sequence and a universal sequence in the sameoligonucleotide. TSU primers described herein include at least one TSUpromoter primer oligonucleotide made up of a 5′ promoter sequence, aninternal first universal sequence (U1) and a 3′ first target specificsequence (TS1) that binds specifically to a target sequence contained ina target nucleic acid. Such compositions may further include at leastone TSU non-promoter primer oligonucleotide made-up of a 5′ seconduniversal sequence (U2) and a 3′ second target specific sequence (TS2)which is different from the TS1. The TSU promoter primer and TSUnon-promoter primer may linked in a complex by using anS-oligonucleotide that links the universal sequences of the TSU primersvia hybridization to complementary terminal sequences of theS-oligonucleotide. The compositions may further include at least oneuniversal promoter primer made up of a 5′ promoter sequence and a 3′ U1sequence, and may also include at least one universal primer made up ofa universal sequence that is substantially identical to that of thesecond universal sequence (U2). These compositions do not require anyparticular sequence be used for any particular component of anoligonucleotide so long as the structural and functional aspects of theoligonucleotides are present in the selected sequences chosen forsynthesis of them.

The invention encompasses isothermal amplification methods that use oneor more of the TSU primers as described herein, which include at leastone TSU promoter primer oligonucleotide made up of a 5′ promotersequence, an internal first universal sequence (U1) and a 3′ firsttarget specific sequence (TS1) that binds specifically to a targetsequence contained in a target nucleic acid. The methods make use ofsteps that bind a TSU primer to the target nucleic acid in a targetcapture step whereby the target nucleic acid with the attached TSUprimer is separated from other mixture components before amplificationis initiated. The isothermal amplification includes a first phase inwhich RNA transcripts are made that include at least one universalsequence or two universal sequences flanking at least one targetspecific sequence. The isothermal amplification includes a second phasein which the RNA transcripts from the first phase are used as templatesby using at least one universal primer and enzymatic in vitro nucleicacid synthesis to make a dsDNA that contains a functional promoter usedto transcribe additional RNA transcripts which are the amplificationproducts that may be further cycled in the isothermal amplificationreaction or used to provide a detectable signal that indicates that thetarget nucleic acid was present in the tested sample.

Methods and compositions are disclosed that are useful for amplifyingtarget nucleic acid sequences in vitro in substantially isothermalconditions to produce amplified sequences that can be detected toindicate the presence of the target nucleic acid in a sample. Themethods and compositions are useful for synthesizing amplified nucleicacids to provide useful information for making diagnoses and/orprognoses of medical conditions, detecting the purity or quality ofenvironmental and/or food samples, or investigating forensic evidence.The methods and compositions are advantageous because they allowsynthesis of a variety of nucleic acids to provide highly sensitiveassays over a wide dynamic range that are relatively rapid andinexpensive to perform, making them suitable for use in high throughputand/or automated systems. The methods and compositions are useful forassays that simultaneously analyze multiple different genetic sequences,i.e., mutliplex amplification systems. Preferred compositions areprovided in kits that include defined assay components that are usefulbecause they allow a user to efficiently perform methods that use thecomponents together in an assay to amplify desired targets.

The disclosed compositions and methods increase the efficiency ofisothermal amplification of nucleic acids, which is particularly usefulin multiplex assays that amplify multiple analytes in a single reactionmixture, e.g., for array-based assays. Multiplex isothermaltranscription based amplification assays are often limited toamplification of about six or fewer analyte targets in a single reactionbecause of primer interactions result in inefficient amplification ofone or more of the targets, which decreases assay sensitivity. Althoughdesign and testing of many different primers and primer combinations mayresult in increased amplification efficiency in multiplex assays, thedisclosed systems minimize primer interactions by use of target-specificprimers in an initial phase of amplification followed by use ofuniversal primers to amplify all of target amplicons in a second phaseof amplification. Thus, amplification efficiency increases while theneed to design and test many individual primers or primer combinationsin multiplex reactions decreases. The disclosed compositions and methodsprovide the advantages that the system can amplify one or many desiredtargets present in a complex mixture, including one or more internalcontrol or internal calibrator targets that provide information that theassay was performed properly or is used to quantitate the results.Besides simplifying multiplex assay design, the disclosed compositionsand methods provide advantages of simplifying both the manufacture ofassay reagents the performance of assay steps a limited number ofreagents are used for each desired target. That is, for each desiredtarget only one or a pair of target-specific universal (TSU) primersunique to the desired target are designed for use in an initialamplification phase, and a subsequent amplification phase uses universalreagents that are used in common for amplification of many targets. TSUprimers include both a target-specific (TS) sequence and a universal (U)sequence in the same oligonucleotide, although TSU primers may includeadditional sequences, such as a promoter sequence. The disclosed methodsare versatile and may be used to detect a single target or multipledifferent targets, all amplified in a single reaction, from whichamplification products may be detected at the end of a reaction(end-point detection) or during the reaction (real-time detection).Typically, the target-specific universal (TSU) primers are provided in atarget capture reagent (TCR) so that the TSU primer is hybridized to anisolated target nucleic acid that is use in an initial phase ofamplification, and universal primers specific for the universalsequences introduced by the TSU primers are used in a subsequentamplification reaction mixture.

Unless otherwise described, scientific and technical terms used hereinhave the same meaning as commonly understood by those skilled in the artof molecular biology based on technical literature, e.g., Dictionary ofMicrobiology and Molecular Biology, 2nd ed. (Singleton et al., 1994,John Wiley & Sons, New York, N.Y.), or other well-known technicalpublications related to molecular biology. Unless otherwise described,techniques employed or contemplated herein are standard methods wellknown in the art of molecular biology. To aid in understanding aspectsof the disclosed methods and compositions, some terms are described inmore detail or illustrated by embodiments described herein.

Nucleic acid refers to a polynucleotide compound, which includesoligonucleotides, comprising nucleosides or nucleoside analogs that havenitrogenous heterocyclic bases or base analogs, covalently linked bystandard phosphodiester bonds or other linkages. Nucleic acids includeRNA, DNA, chimeric DNA-RNA polymers or analogs thereof. In a nucleicacid, the backbone may be made up of a variety of linkages, includingone or more of sugar-phosphodiester linkages, peptide-nucleic acid (PNA)linkages (PCT No. WO 95/32305), phosphorothioate linkages,methylphosphonate linkages, or combinations thereof. Sugar moieties in anucleic acid may be ribose, deoxyribose, or similar compounds withsubstitutions, e.g., 2′ methoxy and 2′ halide (e.g., 2′-F)substitutions. Nitrogenous bases may be conventional bases (A, G, C, T,U), analogs thereof (e.g., inosine; The Biochemistry of the NucleicAcids 5-36, Adams et al., ed., 11^(th) ed., 1992), derivatives of purineor pyrimidine bases (e.g., N⁴-methyl deoxygaunosine, deaza- oraza-purines, deaza- or aza-pyrimidines, pyrimidines or purines withaltered or replacement substituent groups at any of a variety ofchemical positions, e.g., 2-amino-6-methylaminopurine, O⁶-methylguanine,4-thio-pyrimidines, 4-amino-pyrimidines,4-dimethylhydrazine-pyrimidines, and O⁴-alkyl-pyrimidines, orpyrazolo-compounds, such as unsubstituted or 3-substitutedpyrazolo[3,4-d]pyrimidine (e.g. U.S. Pat. Nos. 5,378,825, 6,949,367 andPCT No. WO 93/13121). Nucleic acids may include “abasic” positions inwhich the backbone does not have a nitrogenous base at one or morelocations (U.S. Pat. No. 5,585,481, Arnold et al.), e.g., one or moreabasic positions may form a linker region that joins separateoligonucleotide sequences together. A nucleic acid may comprise onlyconventional sugars, bases, and linkages as found in conventional RNAand DNA, or may include conventional components and substitutions (e.g.,conventional bases linked by a 2′ methoxy backbone, or a polymercontaining a mixture of conventional bases and one or more analogs). Theterm includes “locked nucleic acids” (LNA), which contain one or moreLNA nucleotide monomers with a bicyclic furanose unit locked in a RNAmimicking sugar conformation, which enhances hybridization affinity forcomplementary sequences in ssRNA, ssDNA, or dsDNA (Vester et al., 2004,Biochemistry 43(42):13233-41).

The interchangeable terms “oligonucleotide” and “oligomer” refer tonucleic acid polymers generally made of less than 1,000 nucleotide (nt),including those in a size range having a lower limit of about 2 to 5 ntand an upper limit of about 500 to 900 nt. Preferred oligomers are in asize range having a 5 to 15 nt lower limit and a 50 to 500 nt upperlimit, and particularly preferred embodiments are in a size range havinga 10 to 20 nt lower limit and a 25 to 150 nt upper limit. Preferredoligonucleotides are made synthetically by using any well known in vitrochemical or enzymatic method, and may be purified after synthesis byusing standard methods, e.g., high-performance liquid chromatography(HPLC).

Amplification oligonucleotides include primers and oligonucleotides thatare not extended enzymatically, hybridize to a target nucleic acid, orits complement, and participate in an in vitro nucleic acidamplification reaction in which new nucleic acid strands are synthesizedfrom a template strand by using an end of a primer as an initiationpoint for synthesis, which generally is catalyzed by enzymaticpolymerase activity Amplification oligonucleotides that are extendedenzymatically include primers and promoter-primers which include TSUprimers that contain a target-specific (TS) sequence that is identicalor completely complementary to a sequence contained in an analyte(target) nucleic acid sequence, and a universal (U) sequence that is notcontained in or complementary to an analyte sequence but is introducedto serve as a surrogate or tag for an analyte sequence. The U sequencemay be linked to an analyte or TS sequence and is amplified and/ordetected in place of the analyte sequence to indicate the presence ofone or more analytes in a mixture. Embodiments of TSU primers mayinclude additional sequence information, such a promoter sequence,resulting in a TSU primer referred to as a TSU promoter primer. A TSUprimer that does not include a promoter sequence may be referred to as aTSU non-promoter primer to distinguish it from a TSU promoter primer.Embodiments of amplification oligonucleotides that are generallyreferred to as universal primers (UP) contain a sequence used to amplifya universal or tag sequence that has been linked to an analyte sequenceto serve as a surrogate for the analyte in subsequent assay steps.Universal primers (UP) may contain only a universal sequence and maycontain no analyte-specific sequence, but a UP may also containadditional functional sequences, such as a promoter sequence. Terms suchas “universal non-promoter primer” or “universal promoter primer” may beused to distinguish between different UP types. Amplificationoligonucleotides that are not extended enzymatically typically have achemically or structurally blocked 3′ end that inhibits or prevents themfrom being used to initiate enzymatic polymerization but theseoligonucleotides functionally participate in amplification. Examples ofamplification oligonucleotides that are not extended enzymaticallyinclude TSU promoter provider oligonucleotides and blockeroligonucleotides that bind to a target strand to inhibit or preventstrand extension from a primer to proceed beyond the location on thetarget strand where the blocker oligonucleotide is bound.

Sizes of the amplification oligonucleotides are generally determined bythe function portions that are included in the oligonucleotide.Component portions of a promoter primer or promoter provideroligonucleotide include a promoter sequence specific for a RNApolymerase (RNP). RNP and their corresponding promoter sequences arewell known and may be purified from or made synthetically in vitro byusing materials derived from a variety of sources, e.g., viruses,bacteriophages, fungi, yeast, bacteria, animal, plant or human cells.Examples of RNP and promoters include RNA polymerase III and itspromoter (U.S. Pat. No. 7,241,618, Agami et al.), bacteriophage T7 RNApolymerase and its promoter or mutants thereof (U.S. Pat. No. 7,229,765,Ziman et al. and U.S. Pat. No. 7,078,170, Haydock), RNA polymerase andpromoter from Thermus thermophilus (U.S. Pat. No. 7,186,525, Sakanyan etal.), RNA polymerases from HIV-1 or HCV, and plant directed RNPs (U.S.Pat. No. 7,060,813, Odell et al.). A promoter primer or provideroligonucleotide includes a promoter sequence that is linked functionallyto the chosen RNP. Preferred embodiments of promoter primer or promoterprovider oligonucleotides include a T7 promoter sequence that is usedwith T7 RNP, where the promoter sequence is in the range of 25 to 30 nt,such as a promoter sequence of SEQ ID Nos. 67 or 68 (SEQ ID NO:67,aatttaatacgactcactatagggaga; SEQ ID NO:68, gaaattaatacgactcactatagggaga). Amplification oligonucleotides that include auniversal (U) portion typically include a U sequence in a range of 5 to40 nt, with preferred embodiments in a range of 10 to 25 nt, or 10 to 30nt, or 15 to 30 nt. Amplification oligonucleotides that include a targetspecific (TS) portion typically include a TS sequence in a range of 10to 45 nt, with preferred embodiments in a range of 10 to 35 nt or 20 to30 nt. Amplification oligonucleotides that include multiple U sequencesand/or multiple TS sequences will be in a size range that is determinedby the length of its individual functional sequences, e.g., a promoterprimer or provider oligonucleotide that includes a U sequence and a TSsequence will be the sum of the sizes of the promoter, U and TSsequences, and may optionally include linking nucleotides ornon-nucleotide portions (e.g., abasic linkers). Amplificationoligonucleotides made up of multiple functional components as describedherein may be covalently linked by standard phosphodiester linkages,nucleic acid analog linkages, or non-nucleic acid linkages directlybetween the different functional portions or may be covalently linkedtogether by using additional nucleic acid sequences or non-nucleic(e.g., abasic linkages) compounds that serve as spacers betweenfunctional portions. Some embodiments of amplification oligonucleotidesmay be linked together to form a complex by using non-covalent linkages,such as by using interactions of binding pair members between theoligonucleotides, which includes direct hybridization of complementarysequences contained in two or more oligonucleotides, or via a linkingcomponent to which the individual binding pair member of anoligonucleotide binds (e.g., a binding pair member for eacholigonucleotide attached to a support).

In addition to primers, other amplification oligomers may includeblocked oligonucleotides and promoter provider oligomers (e.g., U.S.Pat. Nos. 5,399,491, 5,554,516 and 5,824,518, Kacian et al.; U.S. Pat.Nos. 4,683,195, 4,683,202 and 4,800,159, Mullis et al., and US2006-0046265 A1, Becker et al.). A blocked oligonucleotide refers to anoligonucleotide that includes a chemical and/or structural modification,usually near or at the 3′ terminus, that prevents or impedes initiationof DNA synthesis from the oligonucleotide by enzymatic means. Examplesof such modifications include use of a 3′2′-dideoxynucleotide base, a 3′non-nucleotide moiety that prevents enzymatic extension, or attachmentof a short sequence in 3′ to 5′ orientation to the oligonucleotide tomake a final oligonucleotide with two 5′ termini (i.e., a first 5′ to 3′oligonucleotide attached to a second, usually shorter, 5′ to 3′oligonucleotide by covalently joining the oligonucleotides at their 3′termini). Another example of a modification is a “cap” made up of asequence that is complementary to at least 3 nt at the 3′-end of theoligonucleotide such that the 5′-terminal base of the cap iscomplementary to the 3′-terminal base of the oligonucleotide. Althoughblocked oligonucleotides are not extended synthetically, they mayparticipate in nucleic acid amplification, e.g., by hybridizing to aspecific location on a nucleic acid template strand to impede synthesisof a complementary strand beyond the position at which the blockedoligonucleotide is bound. A promoter provider oligonucleotide refers toan oligonucleotide that contains a promoter sequence usually on anoligonucleotide that includes a first region that hybridizes to a3′-region of a DNA primer extension product (e.g., a cDNA) to form ahybridization complex between the promoter provider oligonucleotide andthe extension product, and a second region, located 5′ to the firstregion, that is a promoter sequence for an RNA polymerase. By formingthe hybridization complex with the extension product, the promoterprovider oligonucleotide can serve as a template for making a dsDNA thatincludes a functional promoter when the extension product or cDNA isused as a template for further strand synthesis, i.e., by extending anewly synthesized strand made from using the cDNA as a template andusing the promoter sequence of the promoter provider oligonucleotide asa template, a substantially double-stranded structure that contains afunctional promoter is synthesized in vitro.

Amplification of a nucleic acid refers to the process of creating invitro nucleic acid strands that are identical or complementary to acomplete or portion of a target nucleic acid sequence, or a universal ortag sequence that serves as a surrogate for the target nucleic acidsequence, all of which are only made if the target nucleic acid ispresent in a sample. Typically, nucleic acid amplification uses one ormore nucleic acid polymerase and/or transcriptase enzymes to producemultiple copies of a target polynucleotide or fragments thereof, or of asequence complementary to the target polynucleotide or fragmentsthereof, or of a universal or tag sequence that has been introduced intothe amplification system to serve as a surrogate for the targetpolynucleotide, such as in a detection step, to indicate the presence ofthe target polynucleotide at some point in the assay. In vitro nucleicacid amplification techniques are well known and includetranscription-associated amplification methods, such as transcriptionmediated amplification (TMA) or nucleic acid sequence basedamplification (NASBA), and other methods such as the Polymerase ChainReaction (PCR), reverse transcriptase-PCR, replicase mediatedamplification, and the Ligase Chain Reaction (LCR).

To aid in understanding some of the embodiments disclosed herein, theTMA method that has been described in detail previously (e.g., U.S. Pat.Nos. 5,399,491, 5,554,516 and 5,824,518, Kacian et al.) is brieflysummarized. In TMA, a target nucleic acid that contains the sequence tobe amplified is provided as single stranded nucleic acid (e.g., ssRNA orssDNA). Any conventional method of converting a double stranded nucleicacid (e.g., dsDNA) to a single-stranded nucleic acid may be used. Apromoter primer binds specifically to the target nucleic acid at itstarget sequence and a reverse transcriptase (RT) extends the 3′ end ofthe promoter primer using the target strand as a template to create acDNA copy, resulting in a RNA:cDNA duplex. RNase activity (e.g., RNaseHof RT enzyme) digests the RNA of the RNA:cDNA duplex and a second primerbinds specifically to its target sequence in the cDNA, downstream fromthe promoter-primer end. Then RT synthesizes a new DNA strand byextending the 3′ end of the second primer using the cDNA as a templateto create a dsDNA that contains a functional promoter sequence. RNApolymerase specific for the functional promoter initiates transcriptionto produce about 100 to 1000 RNA transcripts (amplified copies oramplicons) of the initial target strand. The second primer bindsspecifically to its target sequence in each amplicon and RT creates acDNA from the amplicon RNA template to produce a RNA:cDNA duplex. RNasedigests the amplicon RNA from the RNA:cDNA duplex and thetarget-specific sequence of the promoter primer binds to itscomplementary sequence in the newly synthesized DNA and RT extends the3′ end of the promoter primer to create a dsDNA that contains afunctional promoter to which the RNA polymerase binds and transcribesadditional amplicons that are complementary to the target strand.Autocatalytic cycles that use these steps repeatedly during the reactionproduce about a billion-fold amplification of the initial targetsequence. Amplicons may be detected during amplification (real-timedetection) or at an end point of the reaction (end-point detection) byusing a probe that binds specifically to a sequence contained in theamplicons. Detection of a signal resulting from the bound probesindicates the presence of the target nucleic acid in the sample.

Another form of transcription associated amplification that uses asingle primer and one or more additional amplification oligomers toamplify nucleic acids in vitro by making transcripts that indicate thepresence of the target nucleic acid has been described in detailpreviously (US 20060046265, Becker et al.). Briefly, this single-primermethod uses a priming oligomer, a promoter oligomer (or promoterprovider oligonucleotide) that is modified to prevent the initiation ofDNA synthesis from its 3′ end and, optionally, a binding molecule (e.g.,a 3′-blocked oligomer) to terminate elongation of a cDNA from the targetstrand. The method synthesizes multiple copies of a target sequence bytreating a target nucleic acid that includes a RNA target sequence with(i) a priming oligonucleotide which hybridizes to the 3′-end of thetarget sequence such that a primer extension reaction can be initiatedtherefrom and (ii) a binding molecule that binds to the target nucleicacid adjacent to or near the 5′-end of the target sequence. The primingoligonucleotide is extended in a primer extension reaction by using aDNA polymerase to give a DNA primer extension product complementary tothe target sequence, in which the DNA primer extension product has a 3′end determined by the binding molecule and which is complementary to the5′-end of the target sequence. The method then separates the DNA primerextension product from the target sequence by using an enzyme whichselectively degrades the target sequence and treats the DNA primerextension product with a promoter oligonucleotide made up of a firstregion that hybridizes to a 3′-region of the DNA primer extensionproduct to form a promoter oligonucleotide:DNA primer extension producthybrid, and a second region that is a promoter for an RNA polymerasewhich is situated 5′ to the first region, wherein the promoteroligonucleotide is modified to prevent the initiation of DNA synthesisfrom the promoter oligonucleotide. The method extends the 3′-end of theDNA primer extension product in the promoter oligonucleotide:DNA primerextension product hybrid to add a sequence complementary to the secondregion of the promoter oligonucleotide, which is used to transcribemultiple RNA products complementary to the DNA primer extension productusing an RNA polymerase which recognizes the promoter and initiatestranscription therefrom. This method produces RNA transcripts that aresubstantially identical to the target sequence.

An embodiment of the one-primer transcription mediated amplificationmethod synthesizes multiple copies of a RNA target sequence byhybridizing to the target RNA a primer at a location in the 3′ portionof the target sequence and a 3′ blocked oligomer (i.e., the bindingmolecule) at a location in the 5′ portion of the target sequence. Thenthe DNA polymerase activity of RT initiates extensions from the 3′ endof the primer to produce a cDNA in a duplex with the template strand (aRNA:cDNA duplex). The 3′ blocked oligomer binds to the target strand ata position adjacent to the intended 5′ end of the sequence to beamplified because the bound 3′ blocked oligomer impedes extension of thecDNA beyond that location. That is, the 3′ end of the cDNA is determinedby the position of the binding molecule because polymerization stopswhen the extension product reaches the blocking molecule bound to thetarget strand. The RNA:cDNA duplex is separated by Rnase activity (RNaseH of RT) that degrades the RNA, although those skilled in the art willappreciate that any form of strand separation may be used. A promoterprovider oligomer includes a 5′ promoter sequence for an RNA polymeraseand a 3′ sequence complementary to a sequence in the 3′ region of thecDNA to which it hybridizes. The promoter provider oligomer has amodified 3′ end that includes a blocking moiety to prevent initiation ofDNA synthesis from the 3′ end of the promoter provider oligomer. In theduplex made of the promoter provider hybridized to the cDNA, the 3′-endof the cDNA is extended by using DNA polymerase activity of RT and thepromoter provider oligomer serves as a template to add a promotersequence to the 3′ end of the cDNA, which creates a functionaldouble-stranded promoter made up of the sequence on the promoterprovider oligomer and the complementary cDNA sequence made from thepromoter provider template. RNA polymerase specific for the promotersequence binds to the functional promoter and transcribes multiple RNAtranscripts that are complementary to the cDNA and substantiallyidentical to the target sequence of the initial target RNA strand. Theresulting amplified RNA can cycle through the process again by bindingthe primer and serving as a template for further cDNA production,ultimately producing many amplicons from the initial target nucleic acidpresent in the sample. Embodiments of the single primer transcriptionassociated amplification method do not require use of the 3′ blockedoligomer that serves as a binding molecule and, if a binding molecule isnot included the cDNA product made from the primer has an indeterminate3′ end, but amplification proceeds substantially the same as describedabove. Due to the nature of this amplification method, it is performedunder substantially isothermal conditions, i.e., without cycles ofraising and lowering incubation temperatures to separate strands orallow hybridization of primers as is used in PCR-based methods.

Detection of the amplified products may be accomplished by using anyknown method. For example, the amplified nucleic acids may be associatedwith a surface that results in a detectable physical change, e.g., anelectrical change. Amplified nucleic acids may be detected in solutionphase or by concentrating them in or on a matrix and detecting labelsassociated with them (e.g., an intercalating agent such as ethidiumbromide or cyber green). Other detection methods use probescomplementary to a sequence in the amplified product and detect thepresence of the probe:product complex, or use a complex of probes toamplify the signal detected from amplified products (e.g., U.S. Pat.Nos. 5,424,413 and 5,451,503, Hogan et al., U.S. Pat. No. 5,849,481,Urdea et al.). Other detection methods use a probe in which signalproduction is linked to the presence of the target sequence because achange in signal results only when the labeled probe binds to amplifiedproduct, such as in a molecular beacon, molecular torch, orhybridization switch probe (e.g., U.S. Pat. Nos. 5,118,801 and5,312,728, Lizardi et al., U.S. Pat. Nos. 5,925,517 and 6,150,097, Tyagiet al., U.S. Pat. Nos. 6,849,412, 6,835,542, 6,534,274, and 6,361,945,Becker et al., US 2006-0068417 A1, Becker et al., and US 2006-0194240A1, Arnold et al.). Such probes typically use a label (e.g.,fluorophore) attached to one end of the probe and an interactingcompound (e.g., quencher) attached to another location of the probe toinhibit signal production from the label when the probe is in oneconformation (“closed”) that indicates it is not hybridized to amplifiedproduct, but a detectable signal is produced when the probe ishybridized to the amplified product which changes its conformation (to“open”). Detection of a signal from directly or indirectly labeledprobes that specifically associate with the amplified product indicatesthe presence of the target nucleic acid that was amplified.

Members of a specific binding pair (or binding partners) are moietiesthat specifically recognize and bind each other. Members may be referredto as a first binding pair member (BPM1) and second binding pair member(BPM2), which represent a variety of moieties that specifically bindtogether. Specific binding pairs are exemplified by a receptor and itsligand, enzyme and its substrate, cofactor or coenzyme, an antibody orFab fragment and its antigen or ligand, a sugar and lectin, biotin andstreptavidin or avidin, a ligand and chelating agent, a protein or aminoacid and its specific binding metal such as histidine and nickel,substantially complementary polynucleotide sequences, which includecompletely or partially complementary sequences, and complementaryhomopolymeric sequences. Specific binding pairs may be naturallyoccurring (e.g., enzyme and substrate), synthetic (e.g., syntheticreceptor and synthetic ligand), or a combination of a naturallyoccurring BPM and a synthetic BPM.

Target capture refers to selectively separating a target nucleic acidfrom other components of a sample mixture, such as cellular fragments,organelles, proteins, lipids, carbohydrates, or other nucleic acids. Atarget capture system may be specific and selectively separate apredetermined target nucleic acid from other sample components, e.g., byusing a sequence specific to the intended target nucleic acid, or it maybe nonspecific and selectively separate a target nucleic acid from othersample components by using other characteristics of the target, e.g., aphysical trait of the target nucleic acid that distinguishes it fromother sample components which do not exhibit that physicalcharacteristic. Preferred target capture methods and compositions havebeen previously described in detail (U.S. Pat. Nos. 6,110,678 and6,534,273, Weisburg et al., and U.S. Ser. No. 11/832,367, Becker etal.). Preferred target capture embodiments use a capture probe insolution phase and an immobilized probe attached to a support to form acomplex with the target nucleic acid and separate the captured targetfrom other components.

A capture probe refers to at least one nucleic acid oligomer that joinsa target nucleic acid and an immobilized probe by using binding pairmembers which may be complementary nucleic acid sequences. One captureprobe embodiment binds nonspecifically to a target nucleic acid andlinks it to a support for separation from the sample, whereas anotherembodiment includes a target specific (TS) sequence that bindsspecifically to a sequence in the target nucleic acid and an immobilizedprobe-binding region that binds to an immobilized probe, e.g., byspecific binding pair interaction. In embodiments in which the TSsequence and immobilized probe-binding region are both nucleic acidsequences, they may be covalently joined or may be on differentoligonucleotides joined by one or more linkers Immobilized probe refersto a moiety attached to a support that joins the capture probe to asupport, directly or indirectly, e.g., by joining members of a specificbinding pair, which includes non-nucleic acid binding (e.g., avidin withbiotin) and nucleic acid sequence hybridization. Immobilized probesinclude an oligonucleotide attached to a support to facilitateseparation of bound target from unbound material, such as other samplecomponents and/or other oligonucleotides included in a target capturereaction mixture. A target capture (TC) complex includes the captureprobe's TS sequence hybridized specifically to a sequence in the targetnucleic acid and the capture probe's immobilized probe-binding regionbound to an immobilized probe on a support.

Support refers to known materials, such as matrices or particlesdispersed in solution, which may be made of nitrocellulose, nylon,glass, polyacrylate, mixed polymers, polystyrene, silane, metal orpolypropylene. Preferred supports are magnetically attractableparticles, e.g., monodisperse magnetic spheres of uniform size±5% toprovide consistent results, to which an immobilized probe is joineddirectly (via covalent linkage, chelation, or ionic interaction), orindirectly (via one or more linkers), to provide stable attachment ofthe immobilized probe to the support in conditions used in the targetcapture reaction.

Separating or purifying refers to removal of one or more components of amixture, such as a sample, from one or more other components in themixture. Sample components include nucleic acids in a generally aqueoussolution phase which may include cellular fragments, proteins,carbohydrates, lipids, and other compounds. Preferred embodimentsseparate or remove at least 70% to 80%, and more preferably about 95%,of the target nucleic acid from other components in the mixture.

Label refers to a molecular moiety or compound that can be detected orlead to a detectable response, which may be joined directly orindirectly to a nucleic acid probe. Direct labeling may use bonds orinteractions to link label and probe, which includes covalent bonds,non-covalent interactions (hydrogen bonds, hydrophobic and ionicinteractions), or chelates or coordination complexes. Indirect labelingmay use a bridging moiety or linker (e.g. antibody, oligomer, or othercompound), which is directly or indirectly labeled, which may amplify asignal. Labels include any detectable moiety, e.g., radionuclide, ligandsuch as biotin or avidin, enzyme, enzyme substrate, reactive group,chromophore (detectable dye, particle, or bead), fluorophore, orluminescent compound (bioluminescent, phosphorescent, orchemiluminescent label). Preferred chemiluminescent labels includeacridinium ester (“AE”) and derivatives thereof (U.S. Pat. Nos.5,656,207, 5,658,737, and 5,639,604). Preferred labels are detectable ina homogeneous assay in which bound labeled probe in a mixture exhibits adetectable change compared to that of unbound labeled probe, e.g.,stability or differential degradation, without requiring physicalseparation of bound from unbound forms (e.g., U.S. Pat. Nos. 5,283,174,5,656,207, and 5,658,737). Methods of synthesizing labels, attachinglabels to nucleic acids, and detecting labels are well known (e.g.,Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd ed. (ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989), Chapt.10; U.S. Pat. Nos. 5,658,737, 5,656,207, 5,547,842, 5,283,174, and4,581,333).

An array refers to multiple components arranged in a two-dimensional orthree-dimensional format to allow similar or identical method steps tobe performed on the components substantially simultaneously. Examples ofarrays are well known and include high-density microarrays or gene chipsthat contain 10 to thousands of oligonucleotides attached to a supportin predetermined configuration. Such arrays allow performance of assaysteps on all the oligonucleotides in different positions under the sameconditions, e.g., hybridization of nucleic acids in a sample applied tothe array or detection of specific sequences.

Sample refers to a specimen that may contain an analyte of interest,e.g., microbe, virus, nucleic acid such as a gene, or componentsthereof, which includes nucleic acid sequences in or derived from ananalyte. Samples may be from any source, such as biological specimens orenvironmental sources. Biological specimens include any tissue ormaterial derived from a living or dead organism that may contain ananalyte or nucleic acid in or derived from an analyte. Examples ofbiological samples include respiratory tissue, exudates (e.g.,bronchoalveolar lavage), biopsy, sputum, peripheral blood, plasma,serum, lymph node, gastrointestinal tissue, feces, urine, or otherfluids, tissues or materials. Examples of environmental samples includewater, ice, soil, slurries, debris, biofilms, airborne particles, andaerosols. Samples may be processed specimens or materials, such asobtained from treating a sample by using filtration, centrifugation,sedimentation, or adherence to a medium, such as matrix or support.Other processing of samples may include treatments to physically ormechanically disrupt tissue, cellular aggregates, or cells to releaseintracellular components that include nucleic acids into a solutionwhich may contain other components, such as enzymes, buffers, salts,detergents and the like.

“Consisting essentially of” is used to mean that additionalcomponent(s), composition(s) or method step(s) that do not materiallychange the basic and novel characteristics of an isothermalamplification method that uses universal sequences and TS sequences asdescribed herein may be included in the compositions or methods. Suchcharacteristics include the structures of TSU oligonucleotides,including complexes of multiple TSU oligonucleotides as described hereinand the ability of the methods to detect one or more analytes or targetnucleic acids in a sample by associating one or more universal sequenceswith the respective target sequences, amplifying in a substantiallyisothermal in vitro condition at least one universal sequence thatserves as a surrogate for an analyte or target nucleic acid, anddetecting a response resulting from amplification of the universalsequence to indicate the presence of at least one analyte in the assayedsample. Any component(s), composition(s), or method step(s) that have amaterial effect on the basic characteristics of the claimed compositionsand/or methods fall outside of this term.

Preferred embodiments of the disclosed methods use aspects of isothermalamplification systems that are generally referred to as transcriptionassociated amplification methods, which have been previously describedin detail (U.S. Pat. Nos. 5,399,491 and 5,554,516, Kacian et al.; U.S.Pat. No. 5,437,990, Burg et al.; PCT Nos. WO 88/01302 and WO 88/10315,Gingeras et al.; U.S. Pat. No. 5,130,238, Malek et al.; U.S. Pat. Nos.4,868,105 and 5,124,246, Urdea et al.; PCT No. WO 95/03430, Ryder etal.; and US 2006-0046265 A1, Becker et al.). Examples includetranscription mediated amplification (TMA) and nucleic acid sequencebased amplification (NASBA). Typically, transcription-associatedamplification uses an RNA polymerase to produce multiple RNA transcriptsfrom a nucleic acid template by using a series of steps that employ anRNA polymerase, a DNA polymerase, deoxyribonucleoside triphosphates,ribonucleoside triphosphates, a template complementary amplificationoligonucleotide that includes a promoter sequence, and optionally one ormore other oligonucleotides, which may serve as primers. Preferreddisclosed embodiments are based on TMA (U.S. Pat. Nos. 5,399,491 and5,554,516) or one-primer transcription-associated amplification (US2006-0046265 A1), although a person of ordinary skill in the art willunderstand that other amplification methods based on polymerase mediatedextension of oligonucleotide sequences may be used with the compositionsand/or method steps described herein.

Methods disclosed herein use three basic steps in a universaltranscription-associated amplification reaction. First, a target capture(TC) step includes hybridizing one or more TSU primers (which may be ina linked complex) to the target nucleic acid and capturing thehybridization complex that includes the target and the primer(s) from amixture which separates the target nucleic acid from other samplecomponents. A target capture mixture may include multiple TSU primers,each type specific for a different target nucleic acid that may bepresent in a sample mixture. During the TC step, only those TSU primersthat are specific for a target nucleic acid that is present in thesample mixture will be bound to a target and carried into the subsequentamplification steps, because TSU primers specific for other targets thatare not present in the sample will remain in solution phase and bediscarded or washed away with other sample components beforeamplification begins using the captured target nucleic acids. Thus,extraneous oligonucleotides that might otherwise result in interferenceor competition for resources during amplification are eliminated beforethe amplification steps begin. The captured target-TSU primer complex isused in an isothermal amplification reaction which is described as afirst phase and a second phase of amplification. In the first phase ofamplification, an initiation step extends the TSU primer attached to thetarget nucleic acid strand by enzymatic in vitro nucleic acid synthesiswhich links a universal sequence region of the TSU primer to an initialamplicon made from the target strand which serves as a template. Forexample, if the target strand is RNA, the TSU primer hybridizes to theRNA and serves as an initiation site for synthesis of the cDNA strandthat includes the U sequence present on the TSU primer. In the secondphase of amplification, subsequent synthetic steps in the reaction usethe initial amplicons, which include the U sequence incorporated intothe product in the initial phase, and amplify the initial and subsequentamplicons by using universal primers that hybridize to the universalsequences and are extended enzymatically by using amplicons astemplates. In some embodiments, two universal sequences are introducedinto the initial amplified products of the isothermal amplificationreaction and those universal sequences are the targets of subsequentamplifications that use primers that contain complementary universalsequences to make more amplicons from the captured target sequence. Inother embodiments, one universal sequence is introduced into the initialamplified products and in the second amplification phase steps, primersinclude one with a universal sequence specific for the introduceduniversal sequence and another target specific primer (TSP) that isspecific for a sequence contained in the target nucleic acid strand or acomplementary strand. In some embodiments, universal primers areprovided in a reagent that is mixed with the captured hybridizationcomplexes that include the target strand and TSU primer, in which thereagent also provides one or more other components used in in vitronucleic acid synthesis (e.g., nucleotide triphosphates, enzymes,cofactors and the like) in the second phase.

Oligonucleotides are disclosed for use preferred embodiments of theuniversal transcription associated amplification methods that include:(1) a target specific capture oligomer (which may be referred to as acapture probe), (2) a target-specific universal (TSU) promoter primer orTSU promoter provider, (3) a target-specific universal (TSU)non-promoter primer, (4) a linker oligonucleotide that may be referredto as an S-oligonucleotide which serves to link TSU primers in a complexthat is hybridized via a portion of one TSU oligonucleotide to thetarget strand, (5) a universal promoter primer (which may be referred toas UP1), and (6) a universal non-promoter primer (which may be referredto as UP2).

In some embodiments, two TSU primers are linked together into a complexthat is then hybridized to a target strand by using hybridization of aTS sequence in a TSU primer to a complementary sequence on the targetstrand. Such linking of TSU primers may be mediated by hybridization ofthe TSU primers to a linking oligonucleotide, which is sometimesreferred to as an S-oligonucleotide due to its serpentine shape when itnon-covalently joins two TSU primers in a three-oligonucleotide complex,in which a first end sequence of the S-oligonucleotide that iscomplementary to and hybridized to part of a first TSU primer and asecond end sequence of the S-oligonucleotide is complementary to andhybridized to part of a second TSU primer. In some embodiments, a TSUpromoter primer sequence may be linked to a TSU non-promoter primersequence without use of a S-oligonucleotide linker. For example, a TSUpromoter primer sequence and TSU non-promoter primer sequence may besynthesized as a single oligonucleotide in which both functionalsequences are covalently linked, either directly or indirectly, such asby using an intervening spacer oligonucleotide sequence or anon-nucleotide covalent linker compound. In other embodiments, the twoTSU oligonucleotide sequences may be synthesized as separateoligonucleotides that are joined covalently by subsequently ligatingthen together directly or indirectly, e.g., by use of a random linkersequence. In embodiments in which multiple TSU oligonucleotides arelinked non-covalently into a complex they may be synthesized as separateoligonucleotides and then joined to a single support, e.g., via bindingpair members attached to the support, or the separate TSUoligonucleotides may contain complementary sequences that are directlyhybridized to link the two functional TSU oligonucleotides into acomplex. For example (shown below in “Embodiment a”), a first TSUoligonucleotide is synthesized to contain, in a 5′ to 3′ orientation, a5′ promoter sequence (P), a middle universal sequence (U1), and a 3′target specific sequence (TS1), and a second TSU oligonucleotide issynthesized to contain a 5′ sequence complementary to the promotersequence (P′), a middle universal sequence (U2), and a 3′ targetspecific sequence (TS2). Alternatively (shown below in “Embodiment b”),the second TSU oligonucleotide may be without the U2 sequence to containa 5′ sequence complementary to the promoter sequence (P′) and a 3′target specific sequence (TS2). When the two TSU oligonucleotides aremixed under hybridization conditions, they form a directly hybridized(DH) complex of TSU oligonucleotides as diagrammed below, where verticallines (|||) indicate the hybridization of the complementary P and P′sequences.

A version of Embodiment a is illustrated schematically in FIG. 17 inwhich the two TSU oligonucleotides are shown in a hybridization complexthat is hybridized to a target strand via the TS1 sequence of a firstTSU primer which is hybridized via the complementary P′ and P sequencesto the second TSU oligonucleotide, which is a TSU promoter provideroligonucleotide with a blocked 3′ end.

Alternatively, two TSU primers may be linked together covalently into acomplex that is then hybridized to a target strand by usinghybridization of a TS sequence in a TSU primer to a complementarysequence on the target strand. FIG. 18 illustrates such an embodiment.This embodiment shows two TSU oligonucleotides joined covalently via anon-nucleotide linker (—C₉—C₉—) to form a complex made up of a TSUpromoter provider that includes a blocked 3′ end, and TS2, U2 andpromoter (P) sequences in a 3′ to 5′ orientation linked to a TSU primerthat includes U1 and TS1 sequences in a 5′ to 3′ orientation. Thiscomplex provides one extendable 3′ terminus in the complex thathybridizes to a target strand via the TS1 sequence of the TSU primer.FIG. 18 also shows, hybridized to the target, a blocker oligonucleotideand a TC probe, hybridized to the target via its TS sequence. Manymethods of making covalently linked primers to make a TSU primer complexare envisioned. For example, coupling after the 2 different oligos(primer and promoter primer or provider) are synthesized by using analdehyde.hydrazine coupling pair. Other coupling pairs may be used, e.g.a carboxyl and an amine, condensed using standard carbodimide chemistry.Alternatives for making covalently linked TSU primer complexes includeconstructing the entire complex on the DNA synthesizer. For example, byusing standard 3′ to 5′ synthesis of a TSU primer, incorporation ofspacers (e.g., non-nucleotide linkers or nucleotide linkers, such aspoly-T), 5′ to 3′ synthesis of the TSU promoter primer or provideroligonucleotide by using reverse polarity phosphoramidites, andfinishing the synthesis by adding a 3′ blocker structure, e.g., a Cadded in 3′ to 5′ orientation. Other alternatives use the same basicstrategy, but start with the TSU T7 promoter primer or provideroligonucleotide and end with the non-promoter TSU primer.

Embodiments of the amplification oligonucleotides may be used in methodsteps in which the TSU oligonucleotides do not form a hybridizationcomplex or covalently linked complex of multiple functional sequenceregions. That is, amplification oligonucleotides may be provided insolution phase as individual oligonucleotides or mixtures ofoligonucleotides in which the individual amplification oligonucleotidesfunction in the method steps without first forming a complex of multipleamplification oligonucleotides independent of the target nucleic acid.

In some embodiments, only one TSU oligonucleotide is used in the initialamplification phase in combination with a target specific primer (TSP)that does not contain a universal (U) sequence. For example, a TSUpromoter primer or TSU promoter provider oligonucleotide may be used incombination with a TS primer, or in another example, a TSU primer may beused in combination with a promoter primer or promoter provideroligonucleotide that does not contain a U sequence. That is, only oneTSU oligonucleotide is used in the initial amplification phase tointroduce a U sequence into an amplicon made during in the initial phaseand a TS primer is used as an initiation point for enzymatic synthesisof the initial complementary strand made from the target strand or toserve as a primer to make a strand complementary to the strand made fromthe target strand. In an embodiment that uses only one TSUoligonucleotide, one universal primer specific for the universalsequence introduced by the TSU oligonucleotide is used in the secondphase of amplification. That is, a single universal sequence serves asthe surrogate or tag sequence for that target during the second phase ofamplification.

In certain embodiments in which the promoter sequence in a TSU promoterprimer or promoter provider oligonucleotide is one recognized by abacteriophage T7 RNA polymerase, the TSU promoter primer or provider maybe referred to as a “TSU T7 primer” or “TSU T7 provider” oligonucleotidewhich may be distinguished from a TSU non-promoter primeroligonucleotide (referred to as a “TSU non-T7 primer”), and a universalprimer (UP1) that includes a T7 promoter sequence may be referred to as“T7-UP1 primer” which is distinguished from a universal primer (UP2)that does not contain a promoter sequence (referred to as a “non-T7-UP2primer”).

Table 1 summarizes various combinations of oligonucleotides that may beused in certain embodiments of universal transcription associatedamplification methods described and illustrated herein. Onlyoligonucleotides used in a target capture step and amplification stepsare listed in Table 1 because amplicons may be detected by a variety ofmeans (e.g., intercalating chemicals), which do not all requireadditional oligonucleotides (e.g., detection probes), but those skilledin the art will appreciate that one or more detection probeoligonucleotides may be used in a complete assay that detects ampliconsmade by these methods. For simplicity, Table 1 uses “TMA” to refer to atranscription mediated amplification method that uses two amplificationoligonucleotides that serve as primers for a single target in an initialphase of amplification (i.e., two oligonucleotides that each have a 3′end that is extended enzymatically), whereas “rTMA” is used to refer toa single-primer transcription mediated amplification method that usesonly one amplification oligonucleotide that serves as a primer (i.e.,has a 3′ end that is extended enzymatically) for each analyte in theinitial phase in which other oligonucleotides included in the reactionare not extended enzymatically (see US 20060046265) in the reaction.

Embodiments of compositions and steps included in amplification methodsdescribed herein are illustrated by the figures.

Referring to FIG. 1, oligonucleotides used in methods disclosed hereinare schematically drawn. At the top, a hybridization complex isillustrated that is made up of a TSU promoter primer linkednon-covalently to the S-oligonucleotide which is linked non-covalentlyto the TSU non-promoter primer. In this complex, the TSU promoter primeris diagramed at the top as including a 5′ promoter sequence (P, solidline), a middle universal sequence, U1 (dashed line), and a 3′target-specific sequence, TS1 (double line). The S-oligonucleotide isshown as an S-shaped curve (dotted line) having a 5′ region thatincludes sequence U1′ that is complementary to the universal sequence U1of the TSU promoter primer and a 3′ region that includes sequence U2′that is complementary to the universal sequence U2 of the TSUnon-promoter primer. The TSU non-promoter primer is diagramed at thebottom of the complex includes a 5′ universal sequence, U2 (dashed line)and a 3′ target-specific sequence, TS2 (double line). Hybridizationbetween the universal sequences of the TSU primers and the complementarysequences of the S-oligonucleotide forms the complex. Under the complexthat contains the TSU primers is shown the target-specific captureoligonucleotide which is diagramed as having a 5′ target-specificregion, TS3 (double line), and a 3′ moiety that is a member of aspecific binding pair (triple line), which in some embodiments is ahomopolymeric nucleic acid sequence. Next is shown the universalpromoter primer (UP1) which is diagramed as having a 5′ promotersequence region (solid line) and a 3′ universal sequence region, U1(dashed line). Next is a diagram of the universal non-promoter primer(UP2) which is shown as a universal sequence, U2 (dashed line).

In preferred embodiments, target capture and amplificationoligonucleotides are provided in a minimum of reagents, to minimize thenumber of addition steps required to perform an assay. In a preferredembodiment, two reagent mixtures are provided as follows. In a firstreagent mixture, referred to as a Target Capture Reagent (TCR), the TSUprimers (e.g., TSU-T7 primer and TSU non-T7 primer) and all cofactorsneeded for their specific attachment to the desired target sequences areincluded (e.g., appropriate salts and buffers for hybridization whenmixed with a sample that contains the target nucleic acids). The TCRalso includes all of the oligonucleotides used in the target capturestep, e.g., a capture probe specific for each desired target or anon-specific capture probe, a support to capture the capture probeattached to the target nucleic acid, and any intermediaryoligonucleotides used in target capture, such as an immobilized probe onthe support. A second reagent mixture, referred to as an AmplificationReagent (AR), provides only one set of universal primers, the universalpromoter primer and the universal non-promoter primer, in addition tocompounds used in in vitro nucleic acid synthesis, e.g., nucleotidetriphosphates (NTPs, dNTPs), salts, buffering agents, enzyme cofactors,and enzyme(s).

In use, the TCR is mixed with a sample that contains the intended targetnucleic acids. The TCR that contains target capture oligonucleotides andTSU primer allows all of the introduced oligonucleotides tosimultaneously hybridize specifically to their respective complementarysequences for each intended target nucleic acid in the sample. Byincluding the TSU primers and the target capture oligonucleotides in thefirst reagent which is mixed with the sample, a complex is formed thatis made up of the target nucleic acid, the TSU primers hybridized to thetarget nucleic acid, and the capture oligonucleotide hybridized to aseparate sequence of the target nucleic acid. Then the complex isattached to the support and separated from other sample components,including primers that are not bound to their intended target nucleicacid, thus limiting the nucleic acids carried into the amplificationstep to the desired targets which are already linked to their specificTSU primers. When the separated complex, attached or detached from thesupport, is mixed with the amplification reagent that contains thecomponents needed for synthesis (e.g., NTPs, salts, buffering agents)and the universal primers, the target nucleic acid is already hybridizedto the TSU primers allowing the initial synthesis to occur to produce aproduct that contains a universal sequence complementary to theuniversal primers (i.e., the universal promoter primer and the universalnon-promoter primer). Then the universal primers may immediatelyhybridize to the complementary universal sequences present in theinitial synthetic products, allowing the amplification reaction tocontinue without an additional step to introduce the universal set ofprimers into the reaction mixture. The universal primers also precludeintroducing into the reaction mixture target-specific sequences whichmay interact with other primer sequences, either intermolecularly orintramolecularly, which can lead to artifacts during subsequentsynthetic steps of the amplification reaction.

An embodiment diagramed in FIG. 2 illustrates the target capture phaseof the universal isothermal amplification method that involves specificbinding of a target nucleic acid in the sample to its respective TSUprimers and to its respective target-specific capture oligonucleotide.FIG. 2, 1. illustrates a target capture reagent (TCR) that is a mixtureof multiple different TSU primer complexes (each containing targetspecific sequences, TSa, TSb, and TSc, which are specific for thedifferent targets, a, b, and c). The TCR also contains thetarget-specific capture oligonucleotides for each of the potentialtargets, with the 3′ member of the binding pair shown as a poly-Asequence. The TSU primer complexes are shown as a TSU promoter primerlinked via an S-oligonucleotide to a TSU non-promoter primer, and thecapture oligomers are shown as a solid line and a poly-A region, bothsubstantially as shown in FIG. 1. For each set of TSU primer complexesand capture oligomers specific for a target nucleic acid, thetarget-specific regions are labeled as TSa, TSb, or TSc. The TCR alsocontains a support with an attached immobilized moiety that bindsspecifically to the capture oligomers (see FIG. 2, 3.). In FIG. 2, 2.,the sample which contains a target nucleic acid (Target a) is mixed withthe TCR, which allows binding of the target specific sequence of the TSacapture probe to bind to its complementary sequence in Target a, and thetarget specific sequence of the promoter primer in the TSU primercomplex to bind to its complementary sequence in Target a. The poly-Asequence of the TSa capture probe binds to its complementary poly-Tsequence of the immobilized probe attached to the support, which allowsthe captured Target a with the TSa TSU primer complex to be retrievedfrom the mixture with the support (see FIG. 2, 3.). The waste productsof the target capture step, following separation of the immobilizedcomplexes on the supports, include the unbound TSU primer complexes(TSUb and TSUc primer complexes, see FIG. 2. 4.), thereby removing themfrom the captured target nucleic acid that is used in a subsequentamplification process.

FIG. 3 illustrates a TSU primer complex, such as shown in FIG. 2 (3.),in more detail. The target strand is in a capture complex made up of thetarget strand, a capture probe that contains a 5′ target specificsequence (TS3) that hybridizes specifically to a complementary targetsequence (TS3′) and a 3′ poly-A sequence, shown hybridized to animmobilized probe that is a complementary poly-T sequence which isattached to a support. Vertical lines (|||||) are used to indicatehybridization between some of the complementary sequence regions. Thetarget strand is also attached to a TSU primer complex by hybridizationbetween the target's TS1′ sequence region and the complementary targetspecific sequence region (TS1) of the TSU promoter primer in the TSUprimer complex. The TSU primer complex is made up of the TSUnon-promoter primer hybridized at its U2 sequence region to thecomplementary U2′ sequence region of the S-oligonucleotide, which has a3′ blocked end (⊖), and the 5′ region of the S-oligonucleotide ishybridized at its U1′ sequence region to the complementary U1 sequenceregion in the TSU promoter primer that includes a 5′ promoter sequenceregion (P) and a 3′ TS1 region. The target strand contains a targetspecific sequence region (TS2) which is identical to the target specificsequence region (TS2) of the TSU non-promoter primer. All of the targetspecific regions of the target strand (TS1′, TS2 and TS3′) areindependent sequences in the target strand.

FIG. 4 illustrates a preferred embodiment of a TSU primer complex,similar to one illustrated in FIG. 3, in which the upper strand is a TSUnon-promoter primer made up of a 3′ TS2 region and a 5′ universalsequence region, U2(+), which is hybridized to a 3′ complementary U2′sequence region of the S-oligonucleotide, which has a 3′ blocked endmade up of a 3′ to 3′ C linkage. The S-oligonucleotide contains anabasic spacer that links the 3′ U2′ sequence region to the 5′ U1′sequence region which is the complement of the U1(−) sequence region inthe TSU promoter primer, to which it is hybridized. The TSU promoterprimer includes a 5′ promoter sequence (P) and a 3′ target specificsequence region (TS1) that flank an internal U1 region. Preferredembodiments of this type of S-oligonucleotide include as the spacer anabasic compound, e.g., (C9)₂ or (C9)₃ that is covalently joined to theflanking U1′ and U2′ sequences.

Although FIG. 2 illustrates only three different TSU primer complexesand capture probes (labeled TSUa, TSUb, and TSUc for Targets a, b and c,respectively) and only one target nucleic acid (Target a), it will beappreciated that many different TSU complexes and captureoligonucleotides, each specific for its own respective target nucleicacid, may be included in a TCR. And a sample may include many differenttarget nucleic acids, all of which may be selectively removed from othersample components. Thus, by including additional TSU primer complexesand probes in a TCR, but using substantially the same steps illustratedin FIG. 2, one or more different targets with attached TSU primers andcapture oligonucleotides each bound specifically to their respectivetargets, may be separated from the mixture by using one or more supportsthat bind to one or more target-primer complexes selectively. Forexample, different size particles may be used as supports, each with adifferent immobilized probe that selectively binds a target specificcapture probe, so that each desired target present in a single samplemay be selectively removed by size separating the supports with theirattached captured target and TSU primer complexes. Although FIG. 2illustrates capture probes that include poly-A regions to hybridize toimmobilized poly-T sequences, those skilled in the art will appreciatethat members of any specific binding pair may be used to capture atarget nucleic acid to a support, and different binding pair members maybe used to selectively isolate different targets from a complex samplemixture. For example, referring to FIG. 2, the TSUa primer complexesspecific to Target a, could be separated from the mixture by using a TSacapture probe that contains a ligand for receptor a in which receptor ais associated with the support as the immobilized probe. And, forexample, Targets a, b, and c all contained in one sample could beassociated with their respective TSU primers and separated from othersample components by using different combinations of binding pairmembers (BPM) on the capture probes (BPMa1, BPMb1, and BPMc1,respectively) which bind to immobilized probes via a specific bindingpair partner (BPMa2, BPMb2, and BPMc2, respectively), to captureindividually the targets, either all to the same support or to supportsspecifically for one or more targets determined by the second bindingpair partner(s) associated with the support(s). For example, a captureprobe for Target a associated with BPMa1 of avidin selectively removesTarget a from the sample by using an immobilized probe having a BPMa2 ofbiotin attached to a first support, whereas in the same TCR, a captureprobe for Target b is associated with a BPMb1 of an Fab fragment whichselectively removed Target b by using an immobilized probe having aBPMa2 of the ligand for the Fab fragment attached to a second support,where the first and second supports are separable by standardmethodologies. Supports with attached complexes that include the desiredtarget nucleic acids may be separated from the other components in themixture, including other sample components, such as cell debris,organelles, proteins, lipids, carbohydrates, other nucleic acids, andfrom unbound primers and capture probes. Any of a variety of well-knownways may be used to separate supports with attached complexes from othercomponents in the mixture, e.g. by centrifugation, filtration, gravityseparation, magnetic separation of magnetized materials, aspiration, andthe like. Thus, following target capture, only TSU primers bound totheir respective targets are carried into the amplification phase of theassay because unbound oligonucleotides are separated from the targetsduring the target capture phase. Additional washing step(s) may beincluded in the target capture phase to wash supports with the attachedtargets and primer complexes, thus further purifying the captured targetnucleic acids with attached TSU primers form other sample components andunbound oligonucleotides before the amplification phase.

Next, amplification is initiated by using the TSU primers specific forthe intended target nucleic acids, i.e., primers carried into theamplification mixture with the captured complex that includes the targetnucleic acid strand linked by hybridization to its corresponding TSUprimer(s). In some preferred embodiments, the TSU primers carried intothe amplification phase are in a TSU primer complex made up of a TSUpromoter primer, S-oligonucleotide, and TSU non-promoter primer for theintended target (see FIG. 1 and FIG. 2). Other TSU primers specific forother analytes that were absent from the sample, and therefore notcaptured, are discarded in the target capture stage and aresubstantially absent from the amplification reaction mixture. Thus, theinitial synthetic step in amplification relies on TSU primers attachedspecifically to the intended target nucleic acids present in at initialamplification phase. Because the TSU primers are already linkedspecifically to their intended target nucleic acid sequences,amplification initiates efficiently when other reaction components(e.g., enzymes and co-factors, synthetic substrates) are mixed with thecaptured target and its attached TSU primer or primer complex. The 3′end of the TSU promoter primer is extended synthetically as illustratedin FIG. 5 which shows the product that results from a first syntheticstep in the initial amplification phase, in which the 3′ end of the TSUpromoter primer, hybridized at its TS1 sequence to the TS1′ sequence ofthe target strand, has been synthetically extended to make a firststrand cDNA. For simplicity, the other components of a TSU primercomplex (the S-oligonucleotide and TSU non-promoter primer) have notbeen illustrated in FIG. 5, but it will be understood that the entireTSU primer complex may be attached to the RNA template strand duringthis synthetic step. Synthesis that initiates from the TSU promoterprimer on the RNA template strand uses an RNA directed DNA polymerase ofa reverse transcriptase (RT) enzyme supplied in the amplificationreaction mixture to synthesize a complementary DNA (cDNA) strand. Apreferred RT is one that includes RNAse H activity to degrade an RNAtarget/template strand, although the RNA dependent DNA polymeraseactivity and the RNA degradation activity may be supplied by differentenzymes in the amplification reaction mixture. The synthesized cDNAstrand contains a sequence TS2′ which is complementary to the TS2sequence in the target/template strand. Following synthesis of the cDNA,degradation of the RNA template strand occurs from the RNAse H activityin the reaction mixture, resulting in a single strand DNA that containsa 5′ promoter sequence, the U1 sequence and the TS1 sequence, allsupplied by the TSU promoter primer, and a 3′ sequence that containssequence complementary to the RNA template strand, including the TS2′sequence which is 3′ of the TS1, U1 and P sequences. This resulting cDNAstrand is shown in FIG. 6.

The first strand cDNA then binds to the TSU non-promoter primer byhybridization between the TS2′ sequence of the cDNA and thecomplementary TS2 sequence of the TSU non-promoter primer, which wascarried into the amplification reaction mixture as part of the TSUprimer complex bound to the captured target nucleic acid. In preferredembodiments, the isothermal amplification conditions maintain the TSUnon-promoter primer in a TSU primer complex (i.e., linked via theS-oligonucleotide to the TSU promoter primer) during the initial cDNAsynthesis step and then the 3′ TS2 portion of the TSU non-promoterprimer in the complex hybridizes to the cDNA strand. Such embodimentsare advantageous because they make use of efficient kinetics ofhybridization that performs substantially as intramolecularhybridization because the TS2 and TS2′ sequences are in close proximitydue to the maintained structure of the TSU primer complex joined to thecDNA. Referring to FIG. 7, the 3′ end of the TSU non-promoter primerhybridized the cDNA strand via hybridization of the TS2 and TS2′sequences is enzymatically extended by a DNA polymerase using the cDNAas a template strand to synthesize a second strand of DNA. Forsimplicity, FIG. 7 shows the TSU non-promoter primer without the othercomponents of the TSU primer complex as described above, but thosecomponents may be maintained during synthesis of the second strand DNA.The second strand DNA includes a 5′ universal sequence (U2) and TS2sequence, both contributed by the TSU non-promoter primer, a DNA strandextended from the 3′ end of the TSU primer, which includes a TS1′sequence and universal sequence U1′ (both complementary to the TS1 andU1 sequences, respectively, of the cDNA and the TSU promoter primer),and a 3′ sequence complementary to the promoter sequence (P) of the TSUpromoter primer. The resulting structure is a substantially dsDNA thatcontains a functional promoter sequence for its respective RNApolymerase enzyme.

Continuing the initial phase of isothermal amplification, as shown inFIG. 8, the RNA polymerase (RNA Pol) specific for the promoter sequencebinds to the functional promoter and initiates transcription from thesubstantially dsDNA, to make multiple RNA transcripts. These transcriptsinclude a 5′ U1 sequence, followed by the TS1 sequence, additionaltarget-specific sequence located between the TS1 and TS2′ sequences, theTS2′ sequence, and a 3′ U2′ sequence. The RNA transcripts contain targetspecific sequences flanked by a first universal sequence (U1), and asecond universal sequence (U2′), which differ from each other (one suchtranscript is illustrated in FIG. 9).

In the second phase of amplification, universal primers (UP1 and UP2 ofFIG. 1) are used to make additional RNA transcripts in a continuouscycle of isothermal amplification, using RNA transcripts as templatesfor synthesis of additional amplification products or amplicons.Preferred embodiments use the universal primers in an isothermalamplification reaction similar to TMA or NASBA reactions. In a firststep of the second phase of amplification, a universal non-promoterprimer (UP2), which consists essentially of a U2 sequence complementaryto the 3′ U2′ sequence of the RNA transcripts produced in the firstphase of amplification, hybridizes to the initial RNA transcripts (seeFIG. 9). The 3′ end of the UP2 primer is extended synthetically in anenzymatic isothermal reaction as illustrated in FIG. 10, in which theRNA transcripts from the initial phase of amplification enter the secondphase at the lower left. The RT enzyme binds and initiates cDNAsynthesis from the 3′ end of the UP2 primer by using the RNA directedDNA polymerase activity and the transcript as a template. Following thedark arrows in FIG. 10 illustrates the steps in the second phase ofamplification. The RNA template strand in the duplex with the cDNA isdegraded by RNAse H activity, allowing the cDNA to hybridize at the U1′sequence to the complementary U1 sequence of the universal promoterprimer (UP1). The RT binds to the 3′ end of the UP1 primer and initiatessecond strand DNA synthesis by using the DNA directed DNA polymeraseactivity and the cDNA strand as a template strand. The resulting dsDNAcontains a functional promoter sequence and, on each strand, twouniversal sequences flanking the target specific sequences. RNApolymerase (RNA Pol) specific for the promoter sequence binds to thefunctional promoter and makes 100 to 1000 transcripts (RNA amplicons)that are identical structurally to the initial RNA transcripts made inthe first phase of amplification. The additional transcripts serve astemplates for more iterations of the process. The RNA transcripts madein the second phase of amplification become available for use in theamplification process when they are made, i.e., no denaturation step isrequired, thus efficiently amplifying the universal and target specificsequences in a continuous isothermal process. RNA transcripts madeduring the second phase of the isothermal amplification process may bedetected during the reaction (i.e., in real time) or at a designated endpoint of the reaction (e.g., a specific time after beginning theamplification reaction or when amplification substantially terminatesdue to exhaustion of substrates present in the reaction).

The RNA amplicons may be detected by using well known detection methodswhich may detect simply an increase in nucleic acid concentration or maydetect selected amplified sequences. For example, detection mayspecifically detect one or more of the universal sequence(s) orsubsequence(s) thereof, or a target specific sequence(s) or asubsequence thereof, or a contiguous sequence that combines portions ofuniversal and target specific sequences. Preferably, a detection stepthat uses a probe for detection of amplicons allows homogeneousdetection, i.e., detection of the hybridized probe without removal ofun-hybridized probe from the mixture (e.g., U.S. Pat. Nos. 5,639,604 and5,283,174, Arnold Jr. et al.). In preferred embodiments that detect theamplified product near or at the end of the second phase ofamplification, a linear probe is used to provide a detectable signalthat indicates hybridization of the probe to the amplified product. Inpreferred embodiments that detect the amplified product in real time,the probe is preferably a probe in which signal production is linked tothe presence of the target sequence, such as a molecular beacon,molecular torch, or hybridization switch probe, that is labeled with areporter moiety that is detected when the probe binds to amplifiedproduct. Such a probe may include a label, e.g., a fluorophore attachedto one end of the probe and an interacting compound, e.g., a quencherattached to another location of the probe to inhibit signal productionfrom the label when the probe is in a “closed” conformation thatindicates it is not hybridized to the amplified product, whereasdetectable signal is produced when the probe is in “open” conformationthat indicates it is hybridized to the amplified product. Various probestructures and methods of using them have been described previously(e.g., U.S. Pat. Nos. 5,118,801 and 5,312,728, Lizardi et al., U.S. Pat.Nos. 5,925,517 and 6,150,097, Tyagi et al., U.S. Pat. Nos. 6,849,412,6,835,542, 6,534,274, and 6,361,945, Becker et al., U.S. Ser. No.11/173,915, Becker et al., and U.S. Ser. No. 60/657,523, Arnold Jr.).

The methods of target capture and amplification that uses at least oneuniversal sequence described herein may be performed in a variety ofdifferent ways. In some preferred embodiments, all of the steps areperformed substantially in a liquid phase, i.e., one in which most orall of the steps occur with the components in the reactions beingpresent in substantially aqueous media. For example, the steps of targetcapture may be performed in a substantially liquid aqueous mixture thatallows hybridization of the capture probe to the target nucleic acid andthe capture probe to an immobilized probe in solution phase by usingimmobilize probes attached to small particles or beads that are mixed orsuspended in the solution phase. Similarly, in some preferredembodiments, all of the amplification steps are performed by having allof the amplification components (e.g., substrates, templates, enzymesand cofactors) in a solution phase for the entire reaction. Thedetection step that detects a signal resulting from the presence ofamplified products may also be performed in a substantially aqueoussolution phase (e.g., as described in U.S. Pat. Nos. 5,639,604 and5,283,174, Arnold Jr. et al.). In other preferred embodiments, one ormore of the steps in an assay that includes target capture,amplification and detection steps may be performed substantiallyattached to a solid phase, such as a support matrix or particle, tocompartmentalize or localize detection of a particular analyte ofinterest. Such embodiments are advantageous because amplificationproducts may be localized, e.g., temporally or spatially, for separatedetection of signals resulting from the presence of one or more selectedanalytes present in a sample. This is particularly useful when a samplemay contain multiple different analytes that are all treated insubstantially the same reagent mixtures during target capture,amplification and/or detection steps, but for which separate detectionof signals resulting from the presence of amplified products for eachanalyte is desired.

Referring to FIG. 11, two preferred embodiments are illustrated thatallow assay steps to be performed attached to a support. Bothembodiments use a combination of TSU primers (TSU promoter primer andTSU non-promoter primer sequences) that are attached via members of aspecific binding pair to a support. The TSU primers in both embodimentsprovide target specific sequences (TS1 and TS2) and universal sequences(U1 and U2) as described earlier in this disclosure. And bothembodiments use universal primers (UP1 and UP2) in the second phase ofamplification as described earlier in this disclosure. In contrast tothe embodiments that use a TSU primer complex that includes anS-oligonucleotide (e.g., as shown in FIG. 3), the TSU primers of thesetwo embodiments are physically linked by being attached to a support. InFIG. 11, Embodiment 1, the TSU promoter primer and TSU non-promoterprimer sequences are linked to a support via a first binding pair member(BPM1) that binds specifically with a second binding pair member (BPM2)attached to the support. This may be accomplished by synthesizing asingle oligonucleotide that contains all of the structural elements ofthe TSU promoter primer and TSU non-promoter primer sequences in theappropriate order (e.g., 3′-TS2-U2-5′-5′-P-U1-TS1-3′) with a BPM1element associated with the synthetic oligonucleotide, or bysynthesizing two oligonucleotides (TSU promoter primer sequence and TSUnon-promoter primer sequence) which are then attached to the same BPM2moiety via a BPM1 moiety associated with the primers. In FIG. 11,Embodiment 2, the TSU promoter primer oligonucleotide and TSUnon-promoter primer oligonucleotide are linked to the same support via afirst binding pair member (BPM1) associated with each primer that bindsspecifically but independently with a second binding pair member (BPM2)attached to the support. In both embodiments, the TSU primers aremaintained in close proximity by being bound to the same support.Because the TS1 sequence of the TSU promoter primer binds with acomplementary sequence in the target nucleic acid strand (TS1′), the TSUprimer may function as a capture probe to selectively bind and separatethe intended target nucleic acid from a sample mixture, by using thesupport to separate the TSU primer-target complex from other samplecomponents. Then, the TSU primer-target complex attached to the supportand mixed with amplification reaction components (e.g., substrates,enzymes, cofactors) serves as a primer-template complex in the initialphase of amplification substantially as described earlier in thisdisclosure except that the support substitutes for the S-oligonucleotidein providing the TSU non-promoter primer in close proximity to the cDNAsynthesized from the initial TSU primer-target complex. The RNAtranscripts from the first phase of amplification then serve astemplates for the second phase of amplification by using the UP1 and UP2universal primers substantially as described in this disclosure(referring to FIG. 10).

The supports in both embodiments shown in FIG. 11 may be used tolocalize the amplification and detection steps, temporally or spatiallyor both for specific analytes of interest. For example, if threedifferent analytes (A1, A2, A3) are present in a sample, the threedifferent target nucleic acids (T-A1, T-A2, T-A3) may be captured in asingle target capture step by using three different TSU primers attachedto different supports or different locations of one support, each TSUprimer specific for its respective analyte by use of different TS1sequences (TS-A1, TS-A2, TS-A3), each specific for one of the targets.Spatial separation of may result, e.g., when a single support is used towhich the TSU primer complexes are attached at different predeterminedloci, such as in an array. Other embodiments that achieve spatialseparation include different wells or containers of a multi-chambereddevice which contain TSU primer complexes in a predetermined pattern ora random pattern, such as achieved by dispensing a known amount ofsolution in which one or more support particles are suspended at apredetermined probability, e.g., a dilution at which an average of oneor fewer individual supports are deposited at a locus on or in a well orchamber. Spatial separation may also be achieved by selectivelyseparating each of the supports into separate chambers or sections of adevice before performing the amplification step by using a physicalcharacteristic of the support to which each of the different TSU primersis attached. For example, TSU primers having different TS1 sequences(TS-A1, TS-A2, TS-A3) may be attached to different particular supportsthat are separable based on size, density, ligand binding capabilities,magnetic properties and the like, so that the different supports withtheir attached TSU primer-target complexes may be spatially separatedbefore performing amplification steps that all use the same reagents,including the same universal primers. The amplified product detected ata particular spatial location in the detecting step indicates whether aparticular analyte was present in the sample, and the cumulativedetection results of all of the locations may indicate that more thanone analyte was present in the sample, and may provide a quantitative orproportional measurement of each analyte present in the sample. Forexample, if an array of 100 chambers is used in which three differentTSU primer-target complexes (i.e., TS-A1, TS-A2, TS-A3 primers) arespatially separated to produce an average of one TSU primer-targetcomplex per locus before performing amplification steps, and thedetection step results in 10 chambers positive for the TS-A1 primer, 30chambers positive for the TS-A2 primer, and 50 chambers positive for theTS-A3 primer, then the results indicate that the sample contained allthree analytes A1, A2 and A3, in a ratio for A1:A2:A3 of 1:3:5.

Similarly, temporal separation may be used to amplify products fromdifferent target nucleic acids and detect the amplified products. Foreither embodiment of FIG. 11, using the model system of three differentanalytes (A1, A2, A3) present in a sample, the three different targetnucleic acids (T-A1, T-A2, T-A3) may be captured in a single targetcapture step by using three different TSU primer complexes attached tosupports, each TSU primer complex specific for its respective analyte byuse of different TS1 sequences (TS-A1, TS-A2, TS-A3). Amplification inthe first and second phases is performed substantially as describedpreviously herein, except that at different times during theamplification a detection measurement is made for each of the amplifiedproducts, e.g., at a first time (T1) for the A1 product, at a secondtime (T2) for the A2 product, and at a third time (T3) for the A3product, which each product results in a different detectable signalsuch as fluorescence at a different wavelength. Thus, positive signalsdetected only at T1 and T3 indicate that the sample contained onlyanalytes A1 and A3, and did not contain A2. In other embodiments,temporal detections may be made at sequential times over an extendedtime range during the amplification reaction, e.g., at T1, T4 and T7 forA1, at T2, T5 and T8 for A2, and at T3, T6 and T9 for A3, and thecumulative results may indicate both the presence and relative amountsof each of the analytes present in a sample. For example if a positivesignal is detected at T1, T4 and T7 it indicates for A1 is present inthe sample, and a positive signal is detected at T8 it indicates that A2is present in the sample, and a positive signal is detected at T6 and T9it indicates that A3 is present in the sample. Amplification for each ofthe analytes is expected to proceed at approximately the same rate dueto use of the same conditions and universal primers in the second phaseof amplification. Thus the relative amount of amplified product and theresulting earliest time of signal detection for each amplified productprovides an indication of the proportional amount of each of theanalytes present in the sample. Based on the model system results abovein which signal for A1 is detected before signal for A3, which isdetected before signal for A2, the relative of amounts of each of theanalytes in the sample are A1 greater than A3 greater than A2.

A combination of spatial and temporal separations may be used in anassay to amplify and selectively detect amplified products from morethan one analyte in a reaction, to allow detection of amplified productsfor an analyte at discrete locations and times. For example, spatialseparation may involve use of an array of TSU primer complexes attachedto a support at predetermined loci combined with temporal separation bydetecting signals at different time points from each or selected groupsof loci to detect amplification products resulting from an amplificationreaction performed on the array. In another embodiment, TSU primercomplexes attached to particulate supports may be suspended in solutionphase of an amplification reaction mixture for some portions of theamplification reaction and then sedimented or attracted to a surface ina random or non-random pattern (spatial separation) for detection ofsignal from the localized amplification products made during otherselected times during the amplification reaction (temporal separation)so that the resulting series of cumulative patterns of detectablesignals provide information on both the presence and relative amounts ofanalyte(s) present in the sample. Those skilled in the art willappreciate that a wide variety of spatial, temporal, and combinedspatial and temporal separations may be used to selectively detectamplification products resulting from amplification reactions thatinclude multiple analytes (i.e., multiplex reactions).

Those skilled in the art will also appreciate that other embodiments areencompassed by the general principles of the assays disclosed herein.That is, assays that include a target capture step to separate a targetnucleic acid from a sample and attach an initial TSU primer to theselected target nucleic acid, followed by an isothermal amplificationreaction that is characterized by two phases, in which the first phaseintroduces universal sequences into products made from the targetnucleic acid, and the second phase uses those universal sequences forfurther production of amplification products, which are detected in thefinal stage of the assay. The target capture step includes attachment ofan initial TSU primer that contains a first universal sequence attachesto the target nucleic acid. The target capture step is followed by aninitial phase of isothermal amplification that uses the initial TSUprimer and a second TSU primer, which contains a second universalsequence, to produce RNA transcripts that contain the first universalsequence and the complementary sequence of the second universalsequence, which flank a target specific sequence. This is followed by asecond phase of isothermal amplification in which the RNA transcriptsmade in the first phase are amplified by using a continuous process ofmaking additional RNA transcripts by using universal primers that bindspecifically to the universal sequences (or their complements)introduced by using the initial TSU and second TSU primers. The finaldetection step detects a signal resulting from the amplified productsmade during the second phase of isothermal amplification to indicatethat the target nucleic acid selected in the target capture step waspresent in the tested sample. These general assay steps may be used witha variety of different primers of different sequences which can bereadily designed by those skilled in the art of molecular biology inview of the general structural features of the primers described herein.

Other embodiments of isothermal amplification methods that use universalsequences may use fewer TSU primers and universal primers compared tothe embodiments described above, while retaining features characteristicof the method such as attachment of a TSU primer to the target nucleicacid during the target capture step and but performing isothermalamplification steps by using a combination of universal and targetspecific primers. For example, an embodiment may using only one initialTSU promoter primer which hybridizes to the target nucleic acid duringthe target capture step and is extended synthetically to introduce asingle universal sequence into the cDNA and later into the RNAtranscripts made during the first phase of isothermal amplification, sothat the second phase of amplification uses only a single universalprimer combined with one or more target specific primers to make theamplification products that are detected to indicate the presence of theanalyte(s) in the tested sample. FIG. 12 illustrates two embodiments(Embodiment 1, upper, and Embodiment 2, lower) to compare difference inthe (A.) target capture (TC) step with initial primer attachment and(B.) primers used in the second phase of amplification. Referring toFIG. 12, Embodiment 1 in the TC step attaches to the target strand a TSUprimer complex that includes both a TSU promoter primer and a TSUnon-promoter primer linked by an S-oligonucleotide as described earlierherein, where the target specific portion of the TSU promoter primerbinds to a complementary sequence in the target strand to link auniversal sequence (U1) to the cDNA that will be made by extending the3′ end of the TSU promoter primer in the first phase of isothermalamplification, as described earlier herein. In contrast, Embodiment 2 inthe TC step attaches to the target strand only a TSU promoter primerwhich is hybridized to via its target specific portion to acomplementary sequence in the target strand to link a U1 sequence to thecDNA that will be made by extending the 3′ end of the TSU promoterprimer, as described above. In Embodiment 1, the first phase ofamplification will continue as described earlier with reference to FIGS.5 to 8, in which the TSU non-promoter primer with its universal sequencewill be used to make the second DNA strand, so that the RNA transcriptsmade in the first phase of amplification will contain two universalsequences. In Embodiment 2, instead of using a TSU non-promoter primer,a target specific non-promoter primer is hybridized to a complementarysequence in the cDNA and extended synthetically to make the secondstrand DNA, so that the RNA transcripts made in the first phase ofamplification contain only one universal sequence. Referring to FIG. 12,B., in the second phase of isothermal amplification for Embodiment 1(upper portion), two universal primers, a universal promoter primer(UP1) and universal non-promoter primer (UP2), are used to make RNAamplicons as described earlier with reference to FIG. 10. In contrast,in Embodiment 2, of FIG. 12, B., the second phase of isothermalamplification uses only one universal promoter primer (UP1) combinedwith a target specific primer (TSP). Referring to FIG. 13, in the secondphase of isothermal amplification, RNA amplicons are made by usingsynthetic steps similar to those described above, but by using the TSP(instead of UP2) to initiate synthesis of the cDNA using the RNAtranscripts as templates (starting at lower left in FIG. 13.). That is,in this embodiment, no U2 or U2′ universal sequences are present in thereaction.

An embodiment that uses a single TSU primer and a target specific primermay be used in assays that make use of the TSU primer attached to asupport, similar to those embodiments described above with reference toFIG. 11. FIG. 14 schematically depicts a TSU promoter primeroligonucleotide made up of a promoter sequence (P), a universal sequence(U1) and a target specific sequence (TS1) which is attached to a supportvia a first binding pair member (BPM1) which binds specifically to asecond binding pair member (BPM2) attached to the support. The TSUpromoter primer is used in the first phase of amplificationsubstantially as described above with reference to FIG. 12 (Embodiment2). For the second phase of amplification, a mixture containing auniversal promoter primer (UP1) and a target specific primer (TSP) isused, as shown in FIG. 14, using the steps as described above anddiagramed in FIG. 13, to amplify the RNA transcripts. In one preferredembodiment, a TSU promoter primer attached to a support (as in FIG. 14)may be used to capture the target nucleic acid strand to which ithybridizes by using its TS1 sequence that is complementary to a sequence(TS1′) in the target strand. Alternatively, an embodiment that uses asingle TSU primer attached to a support may be used in combination witha TC step that uses a capture complex (as in FIG. 12, A.) that includesa support, an immobilized probe and a target specific capture probe, asdescribed in detail previously. In an embodiment that uses a TSUpromoter primer attached to a support as the means for separating thetarget nucleic acid from other sample components, then the TSU promoterprimer serves essentially as the capture probe and as the primer forinitiation of cDNA synthesis when the complex that includes the supportand the TSU promoter primer hybridized to the target strand is mixedwith other amplification reagents. In an embodiment that performs a TCstep that uses a capture complex made up of a capture probe hybridizedto the target strand and bound to the immobilized probe attached to thesupport, then the TSU promoter primer hybridized to the target strandand attached to another support acts as the primer for initiation ofcDNA synthesis when the complex is mixed with other amplificationreagents. In both embodiments, the TSU primer attached to a support maybe used to separate amplification products spatially, temporally, or asa combination of spatial and temporal separation as described above withreference to FIG. 11, except that the second phase of isothermalamplification relies on using a TSP instead of a universal primer (UP2).

Embodiments such as those described with reference to FIGS. 12(Embodiment 2), 13 and 14, that use a TSU promoter primer in combinationwith a target specific primer (TSP) are advantageous in a number ofapplications. For example, in assays for detection of one or morespecies or isolates that share a common target sequence (TS1′) that isconserved among the different targets, a TSP may be included for each ofthe different targets by making the TSP sequence specific for eachtarget. For example, a TS1′ sequence that occurs in 16S or 23S rRNAsequence of many members of a genus (e.g., Mycobacterium) may be used todesign a TSU promoter primer that contains a TS1 sequence that will bindto the target 16S or 23S rRNA from all of the intended targets in thegenus. Then, for each of the intended target species included in thegenus targets (e.g., M. tuberculosis, M. avium, M. abscessus, M.africanum, M. asiaticum, M. avium, M. bovis, M. celatum, M. chelonae, M.flavescens, M. fortuitum, M. gastri, M. gordonae, M. haemophilum, M.intracellulare, M. interjectum, M. intermedium, M. kansasii, M.malmoense, M. marinum, M. non-chromogenicum, M. paratuberculosis, M.phlei, M. scrofulaceum, M. shimodei, M. simiae, M. smegmatis, M.szulgai, M. terrae, M. triviale, M. tuberculosis, M. ulcerans or M.xenopi) a TSP specific for each member is designed and used in theisothermal amplification reaction to make amplified products specificfor each target species, which may be individually detected by usingstandard probe hybridization or size separation methods. In anotherexample, related viral targets, such different human papillomavirus(HPV) types may be detected in a single reaction mixture designing a TSUpromoter primer that binds via its TS1 sequence to a common sequence(TS1′) present in all of the desired HPV types to be detected (e.g., HPVtypes 16, 18, 31, 33, 35, 45, 51, 56, 58, 59 and 68). Thus, the initialcDNA made from the TSU promoter primer will be synthesized for each ofthe intended target HPV types present in the sample using HPV mRNA inthe E6/E7 gene target sequence. Then, for amplification and detection ofindividual HPV types of interest, a TSP is designed for each target(e.g., one each for HPV16 and HPV18) or for a combination of relatedtargets (e.g. one specific for both HPV 16 and HPV18), i.e., each TSPbinds specifically to a sequence of its intended HPV type(s) only. EachTSP specific for its target type is used in the isothermal amplificationreaction to make amplified products specific for the selected targettypes and the amplified products are individually detected by usingstandard methods (hybridization, size separation, sequencing) toidentify the HPV type(s) present in the tested sample. Embodiments suchas these are particularly useful for multiplex reactions, in which morethan one selected target is present in a sample and is amplified toproduce a detectable amplified product that is distinguishable fromother amplified products, so that a signal from each amplified productpresent in the reaction mixture indicates the target analytes that werepresent in the tested sample.

Another application for which embodiments that use a single universalsequence provided by a TSU primer combined with multiple target specificprimers (TSP) are useful is for detecting different forms of relatedgenetic sequences or products. For example, cancers may be correlatedwith the presence of certain genetic translocations or translocationbreakpoints (e.g., chronic myelogenous leukemia (CML) associated withtranslocations between human chromosomes 9 and 22 in the abl gene ofchromosome 9 and the “breakpoint cluster region” or bcr gene ofchromosome 22). To detect different types of translocations, anembodiment of the methods described herein uses a TSU primer in whichthe TS1 sequence is specific for a target sequence in a genetic sequenceor mRNA of one of the translocation members (e.g., abl gene) that iscommon to many different cancer-associated translocations, and thereforecan amplify sequences from many different translocations independent ofthe breakpoint. To amplify and detect specific translocations that areassociated with cancers or have particular prognostic value, a varietyof different TSPs are designed (e.g., different bcr sequences), each onespecific for amplifying a particular sequence associated with acancer-associated translocation, where the amplified sequence may bedetected specifically using standard methods (e.g., probe hybridization,sequencing, or size of amplicon). A sample suspected of containingnucleic acid (DNA or RNA) that has a diagnostic translocation sequenceis then amplified using the TSU promoter primer that amplifies manytranslocations in the target and with the many different TSPs,preferably in a single or a few multiplex reactions, and the amplifiedproducts are detected specifically to provide diagnostic or prognosticinformation based on the particular translocation sequences that areamplified and detected.

Similarly, embodiments that use a single universal sequence provided bya TSU primer and multiple target specific primers (TPS) are useful fordetecting different forms of related genetic sequences that occur indifferent expression products of a gene (e.g., PCA3 gene associated withprostate cancer; see U.S. Pat. No. 7,008,765, Bussemakers et al.). Suchdifferent expression products may result from different splicing eventsin RNA transcripts, where some spiced RNAs are diagnostic of a diseaseor provide prognostic value, such as whether a cancer tissue is benignor malignant. In such embodiments, a TSU promoter primer is designed tocontain a TS1 sequence that is specific for a TS1′ sequence contained inall or many forms of the differentially spliced RNA, and the multipleTSPs are designed to each amplify only one form of the differentiallyspliced RNAs. Following amplification using the TSU promoter primer andthe TSPs, preferably in a single multiplex reaction mixture, theamplified products are detected in a way that distinguishes them toprovide information on the particular form(s) of spliced RNA present inthe tested sample.

Other embodiments that use a single universal sequence provided by a TSUprimer and multiple target specific primers (TPS) are useful fordetecting mutations in genetic sequences that provide diagnostic orprognostic information, such as by detecting the presence of one or moresequences that result in drug resistance. For example, a number of HIV-1mutations are associated with the viral infection being resistant totreatment with particular drugs (e.g., see U.S. Pat. No. 6,582,920, Yanget al.). To detect one or more drug resistance mutations in a singlereaction, the TSU primer is designed to contain a TS1 sequence that iscomplementary to HIV-1 mRNA that is common to HIV-1 strains andisolates, independent of whether the strain or isolate contains a drugresistance mutation. The multiple TSPs are designed to amplify aparticular sequence that contains a mutation associated with drugresistance. In some embodiments the TSPs are specific for the drugresistance mutations themselves, whereas in other embodiments, the TSPsare specific for a sequence that does not contain the drug resistancemutation per se, but which amplifies a product that contains the drugresistance mutation. The TSU promoter primer is used with the multipleTSPs, preferably in a single multiplex reaction, to amplify productsthat provide information on whether a drug resistance mutation waspresent in the nucleic acid of the tested sample. For example, forembodiments in which the TSPs are specific for each of the drugresistance mutations to be detected, the presence or absence of thedistinguishable amplified products indicates which mutations are presentin the tested sample. In other embodiments in which the TSPs arespecific for a sequence that does not contain the drug resistancemutation per se, but which amplifies a product that contains the drugresistance mutation(s), then standard methods of detecting themutation(s) are used, e.g., probe hybridization including on an array,sequencing, or size separation, including mass spectrometry.

Testing of embodiments that use TSU primers, TSU primer complexes anduniversal primers, in the isothermal amplification methods as describedherein has been performed and amplified products have been successfullydetected for viral targets and genetic sequences associated with cancermarkers, such as prostate specific antigen (PSA; U.S. Pat. No.6,551,778, Harvey et al.) and PCA3 sequences.

Those skilled in the art of molecular biology will appreciate that TSUoligonucleotides as described herein do not require any specificsequences to function, so long as the chosen sequences fulfill thefunctional requirements of the TSU oligonucleotide. That is, no singlesequence is required for any functional portion of a TSUoligonucleotide, e.g., no particular primer is required for a TSUpromoter primer or promoter provider, so long as the TSU oligonucleotidecontains sequences for all of the functional portions needed for itsfunction for the embodiment for which it is intended as disclosedherein. Similarly, a TSU primer that does not contain a promotersequence does not require any particular sequence so long as it containsa U sequence and a TS sequence that allows it to function for theembodiment for which it is intended as disclosed herein. Similarly, noparticular sequence is required for an S-oligonucleotide, a covalentlylinked oligonucleotide made up of two TSU oligonucleotide sequences, orfor two TSU oligonucleotides that are directly hybridized to each othervia complementary sequences, so long as the appropriate sequences foreach functional portion are included as described for the embodimentsdisclosed herein. Universal primers similarly do not require aparticular sequence but instead are chosen to contain sequences thatperform with the U sequence(s) chosen for the TSU oligonucleotides asdescribed herein. For example, a universal promoter primer or promoterprovider oligonucleotide contains a promoter sequence and a U sequencethat functions in the methods described herein, where the U sequence ofthe universal primer and the U sequence of the TSU promoteroligonucleotide are usually identical, although a U sequence in theuniversal primer may vary from the U sequence of the TSU oligonucleotideat 1 to 3 nt positions and still perform in the methods disclosedherein. Similarly, the universal primer does not rely on any particularsequence but is selected to be identical to the universal sequence ofthe TSU non-promoter primer with which it is used, but U sequence in theuniversal primer may vary from the U sequence of the TSU primer at 1 to3 nt positions and still function in the disclosed methods. Promotersequences are typically the same in all TSU promoter primers or promoterproviders used in an assay for multiple targets because that simplifiesother reaction components (i.e., a single RNA polymerase is used), butdifferent promoter sequences that function with the same or differentRNA polymerases may be used. Those skilled in the art will appreciatethat many different sequences may be incorporated into TSUoligonucleotides, S-oligonucleotides, and universal primers that fallwithin the scope of the compositions described herein, which thoseskilled in the art of nucleic acid amplification are capable ofselecting based on the descriptions of the structural and functionalfeatures of the oligonucleotides as described herein, wherefunctionality may be demonstrated by using routine testing methods.

Embodiments of the compositions and methods described herein may befurther understood by the examples that follow. Method steps used in theexamples have been described herein and the following informationdescribes typical reagents and conditions used in the methods with moreparticularity. Those skilled in the art of nucleic acid amplificationwill appreciate that other reagents and conditions may be used that willnot substantially affecting the process or results so long as guidanceprovided in the description above is followed. For example, althoughtranscription mediated amplification (TMA) methods are described thatuse a promoter primer or promoter provider oligonucleotide and anon-promoter primer in an initial phase of amplification, other methodsof transcription associated nucleic acid amplification in vitro thatrely on primer extension could be modified to use the TSUoligonucleotides as described herein to make amplified products by usinguniversal primers, i.e., the methods are not limited to TMA-basedembodiments. Those skilled in the art of molecular biology will alsounderstand that the disclosed methods and compositions may be performedmanually or in a system that performs one or more steps (e.g.,pipetting, mixing, incubation, and the like) in an automated device orused in any type of known device (e.g., test tubes, multi-tube unitdevices, multi-well devices such as 96-well microtitre plates, and thelike).

Reagents typically used in the methods described in the examples includethe following. Sample Transport Medium (“STM”) contained 15 mM sodiumphosphate monobasic, 15 mM sodium phosphate dibasic, 1 mM EDTA, 1 mMEGTA, and 3% (w/v) lithium lauryl sulfate (LLS), at pH 6.7. SpecimenDilution Buffer contained 300 mM HEPES, 3% (w/v) LLS, 44 mM LiCl, 120 mMLiOH, 40 mM EDTA, at pH 7.4. Target Capture Reagent (TCR) contained 250mM HEPES, 310 mM lithium hydroxide, 1.88 M lithium chloride, 100 mMEDTA, at pH 6.4, and 250 μg/ml of magnetic particles (1 micron SERA-MAG™MG-CM particles, Seradyn, Inc. Indianapolis, Ind.) with (dT)₁₄ oligomerscovalently bound thereto. TC Wash Solution contained 10 mM HEPES, 150 mMsodium chloride, 6.5 mM sodium hydroxide, 1 mM EDTA, 0.3% (v/v) ethanol,0.02% (w/v) methyl paraben, 0.01% (w/v) propyl paraben, and 0.1% (w/v)sodium lauryl sulfate, at pH 7.5. Probe Reagent contained one or morelabeled detection probes in a solution made up of either (1) 100 mMlithium succinate, 3% (w/v) LLS, 10 mM mercaptoethanesulfonate, and 3%(w/v) polyvinylpyrrolidon, or (2) 100 mM lithium succinate, 0.1% (w/v)LLS, and 10 mM mercaptoethanesulfonate. Hybridization Reagent was either(1) 190 mM succinic acid, 17% (w/v) LLS, 100 mM lithium hydroxide, 3 mMEDTA, and 3 mM EGTA, at pH 5.1, or (2) 100 mM succinic acid, 2% (w/v)LLS, 100 mM lithium hydroxide, 15 mM aldrithiol-2, 1.2 M lithiumchloride, 20 mM EDTA, and 3.0% (v/v) ethanol, at pH 4.7. SelectionReagent used to treat mixtures that use AE-labeled detection probescontained 600 mM boric acid, 182.5 mM sodium hydroxide, 1% (v/v)octoxynol (TRITON® X-100), at pH 8.5, and Detection Reagents used toelicit a chemiluminescent signal from AE-labeled probes included (1)Detect Reagent I made of 1 mM nitric acid and 32 mM hydrogen peroxide,and (2) Detect Reagent II (to neutralize pH) which was 1.5 M NaOH.Amplification reagent was a concentrated mixture that was mixed withother reaction components (target, oligonucleotides) to produce amixture containing 47.6 mM Na-HEPES, 12.5 mM N-acetyl-L-cysteine, 2.5%TRITON™ X-100, 54.8 mM KCl, 23 mM MgCl₂, 3 mM NaOH, 0.35 mM of each dNTP(dATP, dCTP, dGTP, dTTP), 7.06 mM rATP, 1.35 mM rCTP, 1.35 mM UTP, 8.85mM rGTP, 0.26 mM Na₂EDTA, 5% v/v glycerol, 2.9% trehalose, 0.225%ethanol, 0.075% methylparaben, 0.015% propylparaben, and 0.002% PhenolRed, at pH 7.5-7.6. Primers and/or probes may be added to the reactionmixture in the amplification reagent or separate from the amplificationreagent. Enzymes used in amplification reaction mixtures were about 90U/μl of MMLV reverse transcriptase (RT) and about 20 U/μl of T7 RNApolymerase per reaction (where 1 U of RT incorporates 1 nmol of dTTP in10 min at 37° C. using 200-400 micromolar oligo dT-primed polyAtemplate, and 1 U of T7 RNA polymerase incorporates 1 nmol of ATP intoRNA in 1 hr at 37° C. using a T7 promoter in a DNA template).

A typical protocol for TMA reactions that detect results by usinglabeled probes at the end of the amplification reaction follows. The TMAreaction uses substantially the procedures described previously indetail (U.S. Pat. Nos. 5,399,491 and 5,554,516, Kacian et al.). Briefly,a reaction mixture (e.g., 0.08 ml) containing amplification reagent,target nucleic acid, and amplification oligomers (e.g., 15 pmol of eacholigomer per reaction) was mixed, covered with silicon oil (0.2 ml) toprevent evaporation, and incubated for 10 min at 62° C. and then for 5min at 42° C., and then the enzyme reagent (0.025 ml containing reversetranscriptase and T7 RNA polymerase) was added, and reaction mixtureswere incubated for 60 min at 42° C. Following amplification, detectionof the amplified products involved mixing the amplification mixture withan acridinium ester (AE) labeled detection probe oligomer specific forthe amplification product (e.g., 0.1 pmol per reaction in 0.1 ml ofprobe reagent, or an amount previously determined to produce a maximumdetectable signal in an acceptable range, such as up to 2,000,000relative light units (“RLU”) from hybridized labeled probe). Mixtures ofprobe and amplified sequences were incubated to bind the probe to theamplified product and then treated to produce chemiluminescent signalfrom hybridized probes substantially as described (U.S. Pat. Nos.5,283,174 and 5,639,604). Briefly, the probe and amplified productmixtures were incubated for 20 min at 62° C., then cooled at roomtemperature about 5 min and selection reagent (0.25 ml) was added,mixed, incubated 10 min at 62° C. and then at room temperature for 15min to hydrolyze the AE label on unbound probes. Chemiluminescence fromAE on bound probes was produced by adding detect reagent I, incubating,adding detect reagent II, and measuring chemiluminescence in aluminometer (e.g., LEADER®, Gen-Probe Inc., San Diego, Calif.).

A general protocol for TMA reactions that detect results in real timefollows. The assay includes purification of target nucleic acids beforeamplification, amplification, and detection of the amplified productsduring amplification. Target capture is performed substantially aspreviously described in detail (U.S. Pat. Nos. 6,110,678, 6,280,952, and6,534,273, Weisburg et al.). Briefly, samples were prepared to containknown amounts of target RNA (in vitro transcripts (“IVT”) present at apredetermined copy level per sample in a total volume of 0.2 ml of a 1:1(v:v) mixture of water and sample transport medium). Each sample wasmixed with 0.05 ml of TCR that typically contained 5 to 15 pmol oftarget capture oligomer (TCO) specific for the analyte nucleic acid tobe captured (i.e., 3′ target-specific binding region) and a 5′ tailregion (e.g., dT₃A₃₀ sequence) for binding to the immobilized probe(e.g., poly-T oligomers attached to paramagnetic particles; 12.5 μg ofparticles with attached oligomers per reaction), 5 to 15 pmol of TSUprimer and/or complex that includes TSU primer and TSU promoter primeror provider sequence for each analyte (for initial phase ofamplification), and optionally 2 to 5 pmol of blocker oligomer (for rTMAamplification reactions). The mixtures were incubated for 25 to 30 minat 60±1° C. and then for 25 to 30 min at room temperature (20 to 25° C.)to form hybridization complexes through which target nucleic acids werebound to the paramagnetic particles which were the isolated by usingmagnetic separation (e.g., KingFisher96™ magnetic particle processor,Thermo Fisher Scientific, Inc., Waltham, Mass.) and washed one timeusing TC wash solution. Particles were re-suspended in 0.06 to 0.1 ml ofamplification reagent and with amplification oligonucleotides used inthe second phase of amplification (e.g., TS primer, universal primer(s),3′ blocked universal promoter provider). Detection probes (e.g.,molecular beacon or molecular torch probes labeled with a fluorescentlabel compound) may be added with amplification oligonucleotides, orwith addition of enzymes, or following addition of enzymes. Reactionmixtures were covered to prevent evaporation and incubated for 1 to 2minutes at 42±0.5° C. While keeping them at 42±0.5° C., the mixtureswere uncovered and mixed with 0.02 ml of enzyme reagent per mixture,covered again, and incubated for 30 to 90 minutes at 42±0.5° C., duringwhich time fluorescence is measured at regular time intervals (e.g.,every minute) which are referred to as “cycles” for data collection anddisplay, which is typically a graph of detected fluorescence unitsversus time (cycles), from which a time of emergence of signal wasdetermined (i.e., time at which fluorescence signal for a sample becomespositive over a background level, which is usually predetermined for theassay).

EXAMPLE 1 Universal TMA (uTMA) System for Detection of Multiple HPVTypes

This example shows the performance of an embodiment of universalisothermal amplification referred to as “half uTMA”, in a system todetect at least 12 human papillomavirus (HPV) types associated with ahigh risk of developing cervical cancer (high-risk HPV types). Thetarget was either 200 or 1,000 copies/reaction (c/rxn) of a single invitro transcript of the specified HPV type. Target capture,amplification and probe detection by using hybridization protectionassay (HPA) which were all performed substantially as described earlier(U.S. Pat. Nos. 6,110,678 and 6,534,273 for target capture, U.S. Pat.Nos. 5,399,491 and 5,554,516 for TMA, and U.S. Pat. Nos. 5,283,174 and5,639,604 for HPV). The target capture mixture contained in the TCreagent 2 pmol each of target capture oligonucleotides of SEQ ID Nos.28-32. The target capture mixture additionally contained 5 pmol each ofHPV TSU T7 promoter primers of SEQ ID Nos. 1-9. Each of these primerscontained the target-specific region, the sequence of the universal T7primer, and a T7 promoter region. Amplification buffer containedreagents for performing TMA plus 15 pmol each of universal T7 primer ofSEQ ID NO:33 and the TS (target-specific) non-T7 primers of SEQ ID Nos.10-13.

During the target capture step, which includes hybridization at 62° C.,the capture oligonucleotides and TSU T7 promoter primers hybridized totheir specific in vitro transcripts; and all unhybridized primers wereremoved during the wash steps. After target capture, the magnetic beadswith bound complex that includes the target strand and hybridized TSUprimer were mixed with amplification reagent containing primers, RNApolymerase, reverse-transcriptase, dNTPs and NTPs, and then incubated at42° C. for 60 minutes. In the first step of the reaction (initialamplification phase), a cDNA transcription template is created whichincorporates the universal T7 primer region and a HPV target-specificbinding region. Amplification proceeds (in the second phase ofamplification) by using the universal T7 promoter primer and a non-T7primer specific for the target in the reaction. RNA amplicons weredetected by HPA by using a mixture of target-specific acridinium ester(AE)-labeled probes of SEQ ID Nos. 20-27. All probes not hybridized toan amplicon target were hydrolyzed by using the selection reagent duringthe HPA procedure and rendered non-chemiluminescent. Probes that werebound to amplicon target and remained protected from hydrolysis. HPAdetection was performed by using the detection reagents, and theresulting chemiluminescent signals were measured and expressed inrelative light units (RLU).

Table 1 shows RLU signals (average of 3 replicates) obtained for 12high-risk HPV types, 4 low-risk HPV types, and negative reactions inwhich no target was added. A positive reaction was scored for RLUgreater than 20,000. In this example, all high-risk HPV types weredetected successfully at 200 c/rxn, except HPV 45 which was positive at1,000 c/rxn. None of the low-risk HPV types tested gave a positivesignal.

TABLE 1 Group Target Avg RLU 200 c/rxn Avg RLU 1,000 c/rxn A1 HPV 163,125,124 3,335,360 HPV 31 345,676 1,524,821 HPV 35 2,948,726 3,207,962A2 HPV 33 2,571,697 3,924,319 HPV 58 922,123 4,270,230 C1 HPV 18 997,3561,438,953 HPV 45 12,839 579,850 HPV 59 1,950,796 2,521,835 C2 HPV 392,466,025 2,452,492 HPV 68 689,548 1,845,594 D HPV 51 1,571,8341,604,492 HPV 56 1,015,787 775,501 Avg 1 mil c/rxn Avg 10 mil c/rxnLow-risk HPV 6 9,431 9,790 types HPV 11 9,839 9,644 HPV 42 9,805 9,628HPV 43 9,683 9,714 Negative 7,612

EXAMPLE 2 Sensitivity of Universal TMA System for Detection of High-RiskHPV Types

This example shows the performance of an embodiment of universalisothermal amplification referred to as a “full uTMA” in a system thatincludes two universal sequences to detect 12 high-risk HPV virus types.The target was either 200 or 2,000 copies/reaction of a single in vitrotranscript of the specified HPV type. Target capture, amplification andHPA detection steps were all performed substantially as described inExample 1 except that different TSU primer combinations were used. Thetarget capture mixture contained 2 pmol each of TC oligonucleotides ofSEQ ID NOs. 28, 29, 30, 31 and 32. The target capture mixtureadditionally contained S-oligonucleotide TSU primer complexes designedto detect the 12 high-risk HPV types. The TSU primer complexes wereformed by hybridizing 5 pmol of TSU T7 promoter primer with 10 pmol ofS-oligonucleotide of SEQ ID NO:35 and 15 pmol of the corresponding TSUnon-T7 primer. The S-oligonucleotide primer complexes consisted of theS-oligonucleotide of SEQ ID NO:35 in hybridization complexes with thefollowing combinations of TSU T7 promoter primer plus TSU non-T7 primer:SEQ ID Nos. 1 plus 14, SEQ ID Nos. 2 plus 14, SEQ ID Nos. 3 plus 14 (thesame TSU non-T7 primer was used for 3 TSU T7 primers directed to arelated group of HPV types), SEQ ID Nos. 4 plus 15, SEQ ID Nos. 5 plus16, SEQ ID Nos. 6 plus 17, SEQ ID Nos. 7 plus 18, SEQ ID Nos. 8 plus 15,and SEQ ID Nos. 9 plus 15 (the same TSU non-T7 primer was used for bothTSU T7 primers directed to a related group of HPV types). Each TSU T7promoter primer contained the target-specific region, the sequence ofthe universal T7 primer, and a T7 promoter region. Each TSU non-T7primer contained the target-specific region and the sequence of theuniversal non-T7 primer. After each S-oligonucleotide primer complex wasformed separately, they were combined in the target capture mixAmplification buffer contained 15 pmol of universal T7 promoter primerof SEQ ID NO:33 and universal non-T7 primer of SEQ ID NO:34.

During target capture hybridization at 62° C., the captureoligonucleotides and TSU T7 promoter primers of the S-oligonucleotideprimer complexes hybridized to their specific in vitro transcripts; andall un-hybridized primers and S-oligonucleotide primer complexes wereremoved during the wash steps. After target capture, the magnetic beadswith bound target/primer complexes were mixed with amplification reagentcontaining universal primers, RNA polymerase, reverse-transcriptase,dNTPs and NTPs, and then incubated at 42° C. for 60 minutes. In thefirst step of the amplification reaction a cDNA transcription templatewas created which incorporates the universal T7 primer region and auniversal non-T7 primer binding region and then amplification proceededby using the universal T7 and non-T7 primers. RNA amplicons weredetected by HPA as described above using a mixture of target-specificAE-labeled probes of SEQ ID Nos. 20 to 27. All probes not hybridized toan amplicon target were hydrolyzed during the HPA procedure and renderednon-chemiluminescent. Probes that were bound to amplicon target andremained protected. HPA detection was performed as described above, andthe resulting chemiluminescent signal was measured and expressed inrelative light units (RLU).

Table 2 shows signals (average of 3 replicates) obtained for 12high-risk HPV types, and negative reactions with no target added. Apositive reaction was scored for RLU greater than 20,000. In thisexample, all high-risk HPV types were detected successfully at 200c/rxn, except HPV 31 which was positive at 2,000 copies per reaction. Inother experiments (data not shown), low-risk HPV types were notdetected.

TABLE 2 Group Target Avg RLU 200 c/rxn Avg RLU 2000 c/rxn A1 HPV 1632,620 209,397 HPV 31 17,123 84,653 HPV 35 28,542 217,063 A2 HPV 3322,276 797,309 HPV 58 236,932 1,383,602 C1 HPV 18 103,672 964,766 HPV 45324,981 1,329,859 HPV 59 29,254 202,631 C2 HPV 39 100,941 1,376,088 HPV68 162,030 943,088 D HPV 51 241,543 1,132,808 HPV 56 447,408 483,658Negative 10,312

EXAMPLE 3 Detection of HPV RNA from Clinical Samples using a uTMA System

This example shows that the “full uTMA” system as described in example 2is capable of detecting HPV RNA from cervical swab or scraping samplespreserved in alcohol-based liquid media (CYTYC™). The procedure wasperformed as described in Example 2, except that 100 μl of the liquidmedia sample was added to 500 μl of target capture mixture in the targetcapture reaction.

The presence of both high- and low-risk HPV was determined by HPV DNAPCR and visualized as bands following separation by agarose gelelectrophoresis. Identity of any HPV viral RNA present in the sampleswas confirmed by DNA sequencing. Samples that produced greater than20,000 RLU using the full uTMA system, were scored as positive. Table 3shows the correlation between HPV type and full uTMA amplificationresults. Positive PCR that resulted in highly visible bands were scoredas “+”, weak bands as “+/−”, and negative results (no visible band) as“−” (and “nd” means not determined). The full uTMA HPV system used inthis example was not optimized for sensitivity or specificity, butcorrectly scored 29 of 34 cervical samples in this study. Samples 6 and26 were probably not detected because of low amounts of HPV RNA.

TABLE 3 PCR HPV type by Targeted high-risk uTMA Sample # Resultsequencing HPV result 1 + HPV 59 yes + 2 + HPV 16 yes + 3 +/− HPV 66 no− 4 + HPV 61 no − 5 + HPV 18 yes + 6 +/− HPV 18 yes − 7 + HPV 16 yes +8 + mixed yes + 9 + 70 no − 10 + HPV 81 no − 11 + mixed yes + 12 + HPV16 yes + 13 + HPV 33 yes + 14 + HPV 58 yes + 15 + HPV 31 yes + 16 + HPV18 yes + 17 − nd nd − 18 − nd no − 19 + HPV 54 no − 20 − nd no − 21 − ndno − 22 − nd no − 23 + HPV 59 yes + 24 + HPV 16 yes + 25 + HPV 81 no −26 +/− HPV 68 yes − 27 + HPV 68 yes + 28 +/− HPV 53 no − 29 + HPV 16yes + 30 + HPV 62 no ++++ 31 + HPV 58 yes + 32 + HPV 16 yes + 33 + HPV58 yes + 34 + HPV 16 yes −

EXAMPLE 4 Detection of PCA3 RNA in Uniplex and Multiplex Modes usingReverse Standard TMA

In this example, reverse TMA was performed in a standard, i.e.,non-universal, format (RS-TMA). The assay was performed in either theuniplex mode, where the only oligonucleotides required for targetcapture, amplification and detection of PCA3 were included, or themultiplex mode, where oligonucleotides required for target capture,amplification and detection of both PCA3 and PSA were included. Theassay was performed substantially equivalently to the general protocoldescribed above. Specifically, PCA3 in vitro transcript (IVT; SEQ IDNO:62) was spiked into water/STM (1:1) at 10⁶, 10⁴ or 10² copies perreaction. For samples run in the uniplex mode, 5 pmol PCA3 TC probe (SEQID NO:53), 2 pmol PCA3 blocker (SEQ ID NO:51), and 5 pmol of PCA3 Non-T7(NT7) primer (SEQ ID NO:49) were spiked into TCR, and 15 pmol of PCA3Non-T7 (NT7) primer (SEQ ID NO:49), 10 pmol of PCA3 T7 promoter provider(SEQ ID NO:50) and 12 pmol PCA3 molecular torch (SEQ ID NO:52) werespiked into amplification reagent (amounts given here and later in thisand other examples are per reaction, unless indicated otherwise). Forsamples run in the multiplex mode, in addition to the PCA3 oligomerslisted above, 5 pmol PSA TC probe (SEQ ID NO:60), 2 pmol PSA blocker(SEQ ID NO:58) and 5 pmol of PSA NT7 primer (SEQ ID NO:56) were alsospiked into TCR, and 15 pmol of PSA NT7 primer (SEQ ID NO:56), 10 pmolof PSA T7 promoter provider (SEQ ID NO:57) and 12 pmol PSA moleculartorch (SEQ ID NO:59) were spiked into amplification reagent. For eachsample, either 3 or 4 replicates were performed.

After the assay was completed, plots of fluorescence versus time wereprepared for each condition (FIG. 19) and average emergence times weredetermined (Table 4).

TABLE 4 Emergence time (min) PCA3 amount Uniplex Multiplex 10⁶ 8.5 12.510⁴ 11.5 >80 10² 14.5 >80

These results demonstrate that the RS-TMA readily detected PCA3 RNA in auniplex mode. However, in a multiplex mode (PSA specificoligonucleotides present in addition to the PCA3 specificoligonucleotides present in the uniplex mode), detection of PCA3 wasseverely hampered. In fact, 10² and 10⁴ copies of PCA3 were undetectableunder the conditions of the assay. This illustrates the problem thatexists with multiplex amplification reactions known in the art.

These results further demonstrate the ability of RS-TMA to quantitatetarget level, as amount of PCA3 was directly related to the emergencetime. One drawback of the RS-TMA method is the small difference inemergence times between relatively large copy level differences of PCA3(i.e., 3 minutes difference in emergence time between 100-folddifferences in PCA3 copy level). This diminishes the ability of theRS-TMA method to accurately discriminate between small differences(e.g., 3-fold) in copy levels.

EXAMPLE 5 Detection of PCA3 RNA in Uniplex and Multiplex Modes usingReverse Universal (Half) TMA

In this example, reverse TMA was performed in a universal (half) TMAformat (RUh-TMA). In this format, a target-specific universal NT7 primer(TSU NT7) containing a specific target binding region and a universalregion at the 5′ end of the oligonucleotide is bound to target in thetarget capture step. Excess TSU-NT7 is washed away. A TSU-NT7 isincluded in the target capture step for each analyte to be detected in amultiplex assay. In the amplification reaction, a universal NT7 primer(same sequence as the universal sequence of all the TSU-NT7 primers) isadded and is used as the NT7 primer in the amplification of all theanalytes to be detected in a multiplex reaction. Also in theamplification reaction, a target specific T7 promoter provider (TS-T7)is added for each target to be detected in a multiplex assay. Aschematic representation of this format is given in FIG. 15.

The assay was performed substantially equivalently to the protocoldescribed in Example 4 above, with the exceptions described below.Specifically, a PCA3 TSU-NT7 primer (5 pmol; SEQ ID NO:48) and PSATSU-NT7 primer (5 pmol: SEQ ID NO:55) were spiked into TCR instead ofthe PCA3 and PSA TS-NT7 primers, respectively, cited in Example 4.Further, a universal NT7 primer (15 pmol; SEQ ID NO:64) was spiked intothe amplification reaction instead of the PCA3 TS-NT7 primer in theuniplex mode and instead of both the PCA3 and PSA TS-NT7 primers in themultiplex mode. All other conditions were the same as those given inExample 5. After the assay was completed, average emergence times weredetermined (Table 5).

TABLE 5 Emergence time (min) Uniplex Multiplex PCA3 amount RS-TMARUh-TMA RS-TMA RUh-TMA 10⁶ 7.0 8.0 11.5 9.5 10⁴ 10.0 12.0 >80 11.5 10²14.0 17.5 >80 24.0

These results demonstrate that the RUh-TMA format readily detected PCA3RNA. In the uniplex mode, emergence times are somewhat later than thecorresponding emergence times obtained with the RS-TMA format. This isfavorable in relation to quantitation, and helps to solve the problemwith RS-TMA cited in Example 4 (i.e., diminished ability of the RS-TMAmethod to accurately discriminate between small differences (e.g.,3-fold) in copy levels). In the multiplex mode, the interferencesobserved in the RS-TMA system are largely overcome, resulting in readydetection of all levels of PCA3 RNA tested.

EXAMPLE 6 Detection of PCA3 RNA in Uniplex and Multiplex Modes usingReverse Universal (Full) TMA (RUf-TMA) in the S-Oligo Format

In this example, reverse TMA was performed in a universal (full) TMAformat (RUh-TMA). In universal (full) TMA, amplification is initiatedwith a TSU-NT7 and a TSU-T7 provider, and subsequent rounds ofamplification are driven by a universal NT7 primer and a universal T7provider. In order to provide each target with the primer and providerrequired for initiation, yet include only a universal primer andprovider in the amplification reaction, a TSU NT7 primer and a TSU T7provider are joined together, this complex is bound to target in thetarget capture step (via hybridization of the target specific region ofthe TSU-NT7 to the target) and excess complex is washed away. Inamplification, the TSU-NT7 primer is extended, and after digestion ofthe target via RNAse H, the target specific region of the TSU-T7provider that is joined to the TSU-NT7 primer binds to the cDNA andamplification is initiated. Amplification then continues using theuniversal NT7 primer and T7 provider that are in the amplificationreagent.

In the S-oligo mode of RUf-TMA described in this example, the TSU-NT7primer and TSU-T7 provider are joined via hybridization of both to anintervening “S-oligo” as shown schematically in FIG. 16. This S-oligocomplex is pre-formed for each analyte to be included in a multiplexassay, then all are added to TCR in the manner that NT7 primers areadded in the RS- and RUh-TMA formats described above.

The assay in this example was performed substantially equivalently tothe protocol described in Example 4 above, with the exceptions describedbelow. Specifically, the multiplex portion of the assay contained theoligonucleotides required for target capture, universal amplificationand real time detection of not only PCA3 and PSA, but also AMACR. PCA3S-oligo complex was prepared by mixing 5 pmol of PCA3 TSU-NT7 primer(SEQ ID NO:48), 7.5 pmol S-oligo (SEQ ID NO:66) and 10 pmol PCA3 TSU-T7provider (SEQ ID NO:50; in this case, the TS- and TSU-T7 providers areone and the same in water/STM/TCR (1/1/0.5). Further, PSA S-oligocomplex was prepared by mixing 5 pmol of PSA TSU-NT7 primer (SEQ IDNO:55), 7.5 pmol S-oligo (SEQ ID NO:66) and 10 pmol PSA TSU-T7 provider(SEQ ID NO:57). AMACR S-oligo complex was prepared by mixing 5 pmol ofAMACR TSU-NT7 primer (SEQ ID NO:36), 7.5 pmol S-oligo (SEQ ID NO:66) and10 pmol AMACR TSU-T7 provider (SEQ ID NO:37). The mixtures wereincubated at room temperature for 30 minutes to allow the complexes toform. PCA3 and PSA TC probes and blockers were spiked into TCR as inExample 5. Additionally, AMACR TC probe (5 pmol; SEQ ID NO:40) and AMACRblocker (2 pmol; SEQ ID NO:38) were also spiked into TCR. PCA3 and PSAS-oligo complexes (5 pmol each) were spiked into TCR instead of PCA3 andPSA TS-NT7 primers, respectively. AMACR S-oligo complex (5 pmol) wasalso spiked into TCR. PCA3 and PSA molecular torches were spiked intoamplification reagent as in Example 5. Additionally, AMACR moleculartorch (12 pmol; SEQ ID NO:39) was also spiked into amplificationreagent. Universal NT7 primer (15 pmol; SEQ ID NO:64) and universal T7provider (10 pmol; SEQ ID NO:65) were spiked into the amplificationreagent instead of the TS-NT7 primer(s) and TS-T7 provider(s). All otherconditions were the same as those given in Example 4.

After the assay was completed, average emergence times were determined(Table 6).

TABLE 6 Emergence time (min) PCA3 amount Uniplex Multiplex 10⁶ 18.1 20.210⁴ 23.4 25.4 10² 34.5 36.5

These results demonstrate that the RUf-TMA format in the S-oligo modereadily detected PCA3 RNA. In the uniplex mode, emergence times aresignificantly later and the time between different copy levels issignificantly greater than the corresponding values obtained with theRS-TMA format. These features are very favorable in relation toquantitation, and helps to solve the problem with RS-TMA cited inExample 5 (i.e., diminished ability of the RS-TMA method to accuratelydiscriminate between small differences (e.g., 3-fold) in copy levels).In the multiplex mode, the interferences observed in the RS-TMA systemare largely overcome, resulting in ready detection of all levels of PCA3RNA tested.

EXAMPLE 7 Detection of PCA3 RNA in Uniplex and Multiplex Modes

In this example, reverse TMA was performed in a universal (full) TMAformat (RUh-TMA) very similar to that described in Example 6. However,instead of via an S-oligo complex, TSU NT7 primer and TSU T7 providerwere joined together using a Directly Hybridized-oligo (DH-oligo)complex. In this mode, the TSU NT7 primer and TSU T7 provider aredirectly hybridized to one another, with no intervening sequence as inthe S-oligo complex. FIG. 17 depicts an example of a DH-oligo complex,in this case with binding occurring via the T7 promoter region of the T7provider.

The assay in this example was performed substantially equivalently tothe protocol described in Example 6, with the exceptions describedbelow. Specifically, PCA3 DH-oligo complex was prepared by mixing 5 pmolof PCA3 DH-TSU-NT7 primer (SEQ ID NO:54) and 5 pmol PCA3 TSU-T7 provider(SEQ ID NO:50) in water/STM/TCR (1/1/0.5). Further, PSA DH-oligo complexwas prepared by mixing 5 pmol of PSA DH-TSU-NT7 primer (SEQ ID NO:61)and 5 pmol PSA TSU-T7 provider (SEQ ID NO:57). The mixtures wereincubated at room temperature for 30 minutes to allow the complexes toform. TC probes and blockers were spiked into TCR as in Example 6, butPCA3 and PSA DH-oligo complexes (5 pmol each) were spiked into TCRinstead PCA3 and PSA S-oligo complexes, respectively. All otherconditions were the same as those given in Example 6, except that thetotal amplification volume was 0.04 mL instead of 0.08 mL (0.03 mLamplification reagent and 0.01 mL enzyme reagent). After the assay wascompleted, average emergence times were determined (Table 7).

TABLE 7 Emergence time (min) PCA3 amount Uniplex Multiplex 5 × 10⁶ 49.550.5 5 × 10⁵ 43.0 44.0 5 × 10⁴ 36.5 37.5 5 × 10³ 30.0 31.0 5 × 10² 24.524.5

These results demonstrate that the RUf-TMA format in the DH-oligo modereadily detected PCA3 RNA. In the uniplex mode, emergence times aresignificantly later and the time between different copy levels issignificantly greater than the corresponding values obtained with theRS-TMA format. These features are very favorable in relation toquantitation, and helps to solve the problem with RS-TMA cited inExample 4 (i.e., diminished ability of the RS-TMA method to accuratelydiscriminate between small differences (e.g., 3-fold) in copy levels).In the multiplex mode, the interferences observed in the RS-TMA systemare largely overcome, resulting in ready detection of all levels of PCA3RNA tested. Plots of emergence time versus PCA3 copy levels for both theuniplex and multiplex assays yielded excellent correlation factors(uniplex R²=1.000; duplex R²=1.000), demonstrating the quantitativenature of these assays.

EXAMPLE 8 Detection of PCA3 RNA in Uniplex and Multiplex Modes usingReverse Universal (Full) TMA (RUf-TMA) in the CL-Oligo Format

In this example, reverse TMA was performed in a universal (full) TMAformat (RUf-TMA) very similar to that described in Example 6. However,instead of via an S-oligo complex, TSU NT7 primer and TSU T7 providerwere joined together using a covalently linked-oligo (CL-oligo) complex.In this mode, the TSU NT7 primer and TSU T7 provider are covalentlylinked to one another at the 5′ -ends of each oligomer. A variety ofmethods can be utilized to achieve such a linking. An example of onepossible scheme is shown schematically in FIG. 18. In this case, the NT7primer and T7 provider are joined 5′ to 5′ with 2 C9 linkers between the2 oligomers.

The assay in this example was performed substantially equivalently tothe protocol described in Example 6 above, with the exceptions describedbelow. Specifically, the multiplex portion of the assay contained theoligonucleotides required for target capture, universal amplificationand real time detection of not only PCA3 and PSA, but also AMACR andCAP2. CL-oligos for each analyte were prepared generally as follows: NT7primers and T7 providers were synthesized using standard phosphoramiditereagents (Sigma Aldrich), except for those listed below, using anExpedite DNA synthesizer (Applied Biosystems, Foster City, Calif.). TheT7 provider was synthesized with a 5′-aldehyde (specialtyphosphoramidite from SoluLink, San Diego, Calif.) and a reverse polaritydC (specialty Control Pore Glass (CPG) reagent from BiosearchTechnologies). The NT7 primer was synthesized with a 5′ C6 amino linker(Glen-Research). Both oligos underwent cleavage and deprotection usingstandard conditions. A bifunctional spacer was then attached to the NT7primer via incubation with Hydrazine-NHS ester (SoluLink) at roomtemperature for 2 hours in 100 mM phosphate buffer (pH 7.40) containing150 mM NaCl. The reaction mixture was then precipitated with sodiumacetate (pH 5.1) and the pellet was dissolved in 100 mM MOPS buffer (pH4.8) containing a 10% excess of the 5′ aldehyde-modified T7 provider.This mixture was left overnight at room temperature and subsequentlydesalted and purified by PAGE.

SEQ ID numbers of oligonucleotides used to construct the CL-oligocomplexes are in Table 8.

TABLE 8 Oligo Analyte Type SEQ ID No PCA3 TSU NT7 primer 48 TSU T7provider 50 PSA TSU NT7 primer 55 TSU T7 provider 57 AMACR TSU NT7primer 36 TSU T7 provider 37 CAP2 TSU NT7 primer 42 TSU T7 provider 43

PCA3 and PSA TC probes and blockers were spiked into TCR as in Example7, but PCA3 and PSA DH-oligo complexes were replaced with PCA3 and PSACL-oligo complexes (5 pmol each), respectively. Additionally, AMACR TCprobe (5 pmol; SEQ ID NO:40), AMACR blocker (2 pmol, SEQ ID NO:38), CAP2TC probe (5 pmol; SEQ ID NO:46) and CAP2 blocker (2 pmol, SEQ ID NO:44)were also spiked into TCR. Further, in addition to the oligonucleotideslisted in Example 7, AMACR molecular torch (12 pmol; SEQ ID NO:39) andCAP2 molecular torch (12 pmol; SEQ ID NO:45) were also spiked into theamplification reagent. All other conditions were the same as those givenin Example 7. After the assay was completed, average emergence timeswere determined (Table 9).

TABLE 9 Emergence time (min) PCA3 amount Uniplex Multiplex 10⁶ 35.0 35.510⁴ 49.0 48.5 10² 59.0 59.5

These results demonstrate that the RUf-TMA format in the CL-oligo modereadily detected PCA3 RNA. In the uniplex mode, emergence times aresignificantly later and the time between different copy levels issignificantly greater than the corresponding values obtained with theRS-TMA format. These features are very favorable in relation toquantitation, and helps to solve the problem with RS-TMA cited inExample 5 (i.e., diminished ability of the RS-TMA method to accuratelydiscriminate between small differences (e.g., 3-fold) in copy levels).In the multiplex mode (quadruplex in this example), the interferencesobserved in the RS-TMA system are largely overcome, resulting in readydetection of all levels of PCA3 RNA tested.

1-23. (canceled)
 24. A method of amplifying two or more target nucleicacids in a sample comprising the steps of: a. mixing a sample suspectedof containing two or more different target nucleic acids with at leasttwo target specific universal (TSU) complexes, each TSU complex beingmade up of i. a TSU promoter oligonucleotide comprising a 5′ promotersequence, an internal first universal sequence (U1), and a 3′ firsttarget specific sequence (TS1) that binds specifically to a targetsequence contained in one of the at least two target nucleic acids,wherein the TSU promoter oligonucleotide is a TSU promoter provideroligonucleotide that has a blocked 3′ terminus that is incapable ofbeing extended by a polymerase, joined to ii. a TSU non-promoter primeroligonucleotide comprising a 5′ second universal sequence (U2) and a 3′second target specific sequence (TS2) which is different from the TS1and which binds specifically to the amplification product generated bythe TSU promoter oligonucleotide to which it is joined, iii. wherein theTSU promoter primer oligonucleotide is joined to the TSU non-promoterprimer oligonucleotide: (A) via a covalent linkage that is apolynucleotide linker sequence, a non-nucleotide abasic linker compoundor a linker containing both nucleotide and non-nucleotide residues, (B)via a hybridization complex between the 5′ promoter sequence and asequence on the TSU non-promoter primer that is complementary to the 5′promoter sequence, or (C) via a hybridization complex that includes anS-oligonucleotide that contains a first sequence complementary to asequence in the TSU promoter oligonucleotide and a second sequencecomplementary to a sequence in the TSU non-promoter primeroligonucleotide, b. hybridizing the various TSU non-promoter primeroligonucleotides of the TSU complexes to target sequences in the targetnucleic acids via the TS2 sequence in the TSU non-promoter primers, c.optionally hybridizing a blocker oligonucleotide to a sequence on thetarget nucleic acids, wherein said blocker comprises a blocked 3′terminus to prevent extension of the blocker by a polymerase and whereinthe blocker oligomer is hybridized to the target nucleic acid at aposition to terminate polymerase extension of the TSU non-promoterprimer, d. isolating the target nucleic acids with hybridized TSUcomplexes away from unhybridized TSU primer complexes and other samplecomponents, e. synthetically extending the 3′ terminus of the TSUnon-promoter primers hybridized to the target nucleic acid by using apolymerase in vitro nucleic acid synthesis in which the target nucleicacid is a template to make a first cDNA strand, f. hybridizing the TSUpromoter provider oligonucleotide of the TSU complex to the respectivefirst cDNA strand by specific hybridization of the TS1 sequence in theTSU promoter provider oligonucleotide to a target sequence contained inthe first cDNA strand, g. synthetically extending the 3′ terminus of thefirst cDNA by using the TSU promoter provider as a template to make asubstantially double-stranded DNA that contains a functional promotersequence and the U1 sequence, h. enzymatically transcribing RNAtranscripts from the functional promoter sequence to make RNAtranscripts that contain a 5′ U1 region sequence, a TS1 sequence, acomplement of the second target specific sequence (TS2′), and a 3′universal sequence that is complementary to the U2 sequence (U2′), i.hybridizing a universal primer oligonucleotide (UP2) that contains auniversal sequence U2 to the RNA transcript at the U2′ sequence, j.synthetically extending the 3′ terminus of the UP2 by enzymatic in vitronucleic acid synthesis to make a cDNA strand, k. hybridizing to the U1′sequence a universal promoter oligonucleotide (UP1) that contains apromoter sequence, a universal sequence U1, and a 3′ blocked end to thecDNA made in step j, l. synthetically extending the 3′ terminus of theDNA strand made in step j using the UP1 oligonucleotide as a template tomake a functional double-stranded promoter using enzymatic in vitronucleic acid synthesis, and m. transcribing multiple RNA transcriptsfrom the functional promoter, which transcripts are amplificationproducts that may serve as templates for further enzymatic in vitronucleic acid synthesis.
 25. The method of claim 24, further comprising adetection step to indicate the presence or absence of each of the two ormore target nucleic acids suspected of being present in the sample. 26.The method of claim 25, wherein the detection method step is selectedfrom the group consisting of hybridizing a detection probe oligomer toan amplification product, determining the size of an amplificationproduct, and sequencing an amplification product.
 27. The method ofclaim 26, wherein the detection probe oligomer is present in thereaction of step h.
 28. The method of claim 26, wherein the detectionprobe oligomer is present in the reaction of step e.
 29. The method ofclaim 25, wherein the detection probe oligomer allows homogeneousdetection of the amplification product.
 30. The method of claim 25,wherein the detection step is performed in real time.
 31. The method ofclaim 30, wherein the detection probe oligomer comprises a fluorophoreattached to one end of the detection probe oligomer and a quencherattached to another location of the detection probe oligomer to inhibitsignal production from the fluorophore when the detection probe oligomeris in a closed conformation, indicating that the detection probeoligomer is not hybridized to the amplification product, but adetectable signal is produced when the detection probe oligomer changesto an open conformation upon hybridizing to the amplification product.32. The method of claim 31, wherein the detection step is performedusing a molecular beacon or a molecular torch.
 33. The method of claim25, wherein the detection step is a quantitative detection step todetermine the amount of the analyte.
 34. The method of claim 24, whereinthe sample is suspected of containing two or more different targetnucleic acids selected from the group consisting of: PCA3, PSA, prostatecancer markers, cancer markers, AMACR, CAP2, RNA with genetictranslocations, RNA with translocation breakpoints, DNA with genetictranslocations, DNA with translocation breakpoints, genetictranslocations that include all or part of the abl gene and all or partof another gene, genetic translocations that include all or part of thebcr gene and all or part of another gene, and bcr-abl.
 35. The method ofclaim 24, wherein the sample is suspected of containing two or moredifferent target nucleic acids selected from the group consisting of HPV16, HPV 18, HPV 31, HPV 33, HPV 35, HPV 39, HPV 45, HPV 51, HPV 56, HPV58, HPV 59, HPV 61, HPV 62, HPV 66, HPV 68, HPV 70, and HPV
 81. 36. Themethod of claim 24, wherein step d comprises hybridizing to each of thetarget nucleic acids a target capture oligonucleotide that contains atarget specific sequence (TS3) that hybridizes specifically to asequence in the target nucleic acid that is different from the sequencein the target nucleic acid that hybridizes to the TS1 sequence of theTSU promoter oligonucleotide or the TS2 sequence of the TSU non-promoterprimer, and contains an immobilized probe-binding region that binds toan immobilized probe attached to a solid support, wherein theimmobilized probe-binding region binds to the immobilized probe by aspecific binding pair interaction; binding the immobilized probe-bindingregions of the target capture oligonucleotides to the immobilized probe,thereby immobilizing the target nucleic acids with hybridized TSUcomplexes to the solid support; and separating the support fromunhybridized TSU primer complexes and other sample components.
 37. Themethod of claim 36, wherein the immobilized probe-binding region is anucleic acid sequence.
 38. The method of claim 37, wherein theimmobilized probe-binding region comprises a poly-A sequence, whereinsaid poly-A sequence is attached to the 3′ end of the target specificcapture oligonucleotide's target specific sequence, and wherein theimmobilized probe comprises a poly-T sequence.
 39. The method of claim24, wherein the TSU promoter oligonucleotide is directly joined to theTSU non-promoter primer oligonucleotide via the covalent linkage. 40.The method of claim 24, wherein the TSU promoter oligonucleotide isdirectly joined to the TSU non-promoter primer oligonucleotide via thehybridization complex between the 5′ promoter sequence and a sequence onthe TSU non-promoter primer that is complementary to the 5′ promotersequence.
 41. The method of claim 24, wherein the TSU promoteroligonucleotide is joined to the TSU non-promoter primer oligonucleotidevia the hybridization complex that includes the S-oligonucleotide. 42.The method of claim 24, wherein the TSU promoter oligonucleotide's 5′promoter sequence is a T7 promoter sequence.
 44. The method of claim 42,wherein the T7 promoter sequence is a promoter sequence of SEQ ID NO:67or SEQ ID NO:68.